CYP2D6 genetic test profiling and the challenges associated with using standard pharmacogenetics panels in a diverse South Florida population.This study focuses on the implementation of CYP2D6 panel including 22 alleles. Phenotype, genotype, and allelic frequencies were compared among different racial and ethnic groups.A total of 413 participants were recruited to participate in this study through Nicklaus Children's Hospital. Buccal swabs were collected and tested using an extended CYP2D6*4, *17, *41, and *2A, showed a statistically significant difference between White Hispanics and Black Non‐Hispanics. Aggregate frequency of all alleles with decreased function varied between 2.8% and 50.0% in different racial and ethnic groups. Additionally, rare allele combinations were observed in this South Florida cohort.The majority of participants (75.0%) self‐identified as Hispanics. Four alleles, CYP2D6 allele frequencies in the distinct racial and ethnic populations of South Florida, demonstrating a unique genetic makeup within South Florida. However, overall, the frequencies of Poor Metabolizer, Normal Metabolizer, Intermediate Metabolizer, and Ultrarapid Metabolizer did not differ between racial and ethnic groups at a statistically significant level.The heterogeneity among Hispanic groups demonstrated in previous literature and by this study reflects the complexity of ethnicity and suggests that a more granular categorization is needed, one based on ancestry and migration history rather than primary language. Overall, we have determined that there are statistically significant differences in  An UM p) Crews, .CYP2D6 can differ from person‐to‐person depending on an individual's CYP2D6 genotype and phenotype. One example is codeine, an opioid analgesic indicated to relieve mild to moderate pain through codeine metabolism and morphine activation by CYP2D6 enzymes . Most participants enrolled at the end of their visit with the neurology, plastic surgery, or hematology/oncology departments. Patients and family members attending the hospital's Cancer Center Mini Relay for Life were also introduced to the study and invited to participate. All patients had to be between the ages of 1–21 years and reside in the South Florida area to be eligible for the study.Patients of Nicklaus Children's Hospital were recruited to participate in this study (® Corporation (Genelex Corporation) using an extended CYP2D6  panel with enough sensitivity to capture over 93.0%–97.0% of the low‐frequency PM phenotypes. Genelex® was selected based on several factors including clinical decision support, turnaround time, breadth of the panel, and cost. Twenty‐two alleles were included in the panel: CYP2D6 *1, *2, *2A, *3, *4, *5, *6, *7, *8, *9, *10, *11, *12, *14A, *15, *17, *19, *20, *29, *35, *36, and *41.Participants were asked to provide their race as either White, Black, Asian, or Mixed, and categorized their ethnicity as Hispanic or Non‐Hispanic. There were 37 participants (8.9%) who refused to provide this information. These patients were included in the analysis of total frequencies of phenotypes. Buccal swabs were collected for DNA extraction. All samples were genotyped at the GenelexAll participants and their primary caregivers if requested were mailed a copy of their individual phenotype/genotype results. The patients were also offered a consultation with a genetic counselor if they wanted a detailed explanation of the study results.p ± 1.96 × SE, where SE = sqrt(p × (1 − p)/n); p is the proportion of an allelic variant or a phenotype in a given race and ethnicity group; n is the total number of patients in the same group. If 95% CI did not overlap, the difference was determined to be statistically significant. All statistical analysis was performed in R. Of note, the interpretation of activity scores by Genelex® for PMs, IMs, NMs, and UMs differed from that of Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. CPIC definitions were used in this study, namely, activity scores of 1.0–2.0 were considered NM.Subsequently, genotype results were analyzed. Allelic frequencies were assessed for statistically significant differences at a 95% confidence interval (CI) for proportions. CI was calculated as 3CYP2D6 alleles examined, 14 were identified in the South Florida population: CYP2D6*1, *2, *2A, *3, *4, *5, *6, *9, *10, *17, *29, *35, *36, and *41. Eight alleles from the panel were not present: CYP2D6*7, *8, *11, *12, *14A, *15, *19, and *20.Of the 413 individuals enrolled in the study, 376 provided information on their race and ethnicity, 75.0% identified as Hispanic, and 19.0%, 2.0%, and 11.0% identified their race as Black, Asian, and Mixed, respectively  than in BNH (2.4%). As a result, PM genotype combinations were only detected in individuals self‐identifying as White.In total, 10 SNPs and 10 deletions/duplications were detected, showing variable allele frequencies among different race/ethnic groups Figure . There wA Figure . The occCYP2D6*17 (15.3% vs. 2.8%), while CYP2D6*41 showed a significant difference in the opposite direction (0.8% vs. 7.5%).  than in the BNH (6.5%).Furthermore, in our study, the normal function CYP2D6*1, *2A, and two *2‐like alleles. Since no deleterious mutations were detected, it is assumed that all four alleles are active, and the phenotype would be expected to be UM. In addition, there was one BNH (0.8%) and one WH (0.2%) study participant with the rare tandem allele *36‐10.Finally, a rare allele combination and a unique duplication were observed in this South Florida cohort. Specifically, we found a BNH individual with four copies of normal function including CYP2D6 allele frequencies in the distinct racial and ethnic populations of South Florida, demonstrating a unique genetic makeup within South Florida. However, overall, frequencies of PM, NM, IM, and UM did not differ between racial and ethnic groups at a statistically significant level , but not in BNH (2.4%), aligning with other studies on WH participants from Cuba, Nicaragua, and Venezuela   compared to WHs (2.8%). These findings also align with reports from other studies on similar populations . Meanwhile, none of the Nicaraguan Mestizos had *17, but 2.7% of the White Cuban population did    Cai, . NotablyKennedy, . Becausey Blake, , 2008.CYP2D6*36‐10 allele in a WH participant. The tandem allele *36‐10 is prevalent in Japanese populations (24.2%–26.7%), but rarely detected among African‐Americans (0.4%), and not reported in White populations   Bernard, . The hetThis study has a few limitations. WH and BNH were the two largest population groups in the study. It is therefore not surprising that the only statistically significant differences found were between the two ethnic groups with the largest sample sizes. It is possible that there are additional frequency differences among other groups in this population, but this study is underpowered to detect them. In addition, the race and ethnicity used in this study were based on self‐reported records by the study participants. These records may be imprecise and biased. Future studies should include design and resources to allow for genetic confirmation or verification of perceived ancestry.CYP2D6 function. For example, CYP2D6*31, *49, *50, *54, *55, *59, and *84 all translate to decreased or no function, however, they were not tested in this study and are not included in most commercial panels. It is currently not known whether these alleles are prevalent in Hispanics or Latinos. If a patient had any of these alleles, they would be incorrectly classified as NM. That could explain the markedly lower rates of PM previously reported in Hispanics (Bernard, CYP2D6 gene, rather than selected polymorphisms. As a whole, sequencing would also provide a better depiction of the allelic variation within underrepresented communities (Barron et al., Overall, our findings suggest that in predominantly Hispanic or Latino populations, the assumption that allelic and phenotype distributions are similar to less diverse populations may be inaccurate. As a result, pharmacogenetic panels designed for less diverse populations may not be appropriate for Hispanic populations. If the subject of a test has an allele that was not included in the panel, they risk improper classification of their Bernard, . Thus, pNone declared.


Context:Carica papaya Linn. (Caricaceae) leaf (CPL) juice has long been traditionally used in ethnomedicine for dengue fever.Objective: The study examines the effects of standardized CPL aqueous extract (SCPLE) on platelet count, extramedullary haematopoiesis (EMH), and immunomodulation in cyclophosphamide (CP)-induced animal model of thrombocytopenia.Materials and methods: The extract was analyzed for myricetin, caffeic acid, trans-ferulic acid, and kaempferol using HPTLC for standardization followed by UPLC-qTOF/MS fingerprinting for metabolite signature. The effects of SCPLE (50 and 150 mg/kg p.o.) on proliferative response of platelet count and total leucocyte count (TLC) were observed up to 14 days in Wistar rat. However, delayed-type hypersensitivity (DTH), haemagglutination titre (HT), and in vivo carbon clearance were examined as immunomodulatory parameters in albino mice at 150 mg/kg p.o. against CP.Results: The quantitative HPTLC estimation of SCPLE showed the presence of myricetin, caffeic acid, trans-ferulic acid, and kaempferol up to 280.16 ± 5.99, 370.18 ± 6.27, 1110.86 ± 2.97, and 160.53 ± 2.48 (μg/g), respectively. Twenty-four metabolites were identified using UPLC-qTOF/MS. Oral administration of SCPLE (150 mg/kg) in thrombocytopenic rats exhibited significant (p < 0.01) increase in thrombocytes (1014.83 × 103 cells/mm3), DTH response (0.16 ± 0.004), and phagocytic index (63.15% increase) as compared to CP-induced thrombocytopenia group. Histopathological studies showed minimal fibrosis in spleen histology.Discussion and conclusions: Results suggest CPL can mediate the release of platelets providing the means for the treatment and prevention of dengue. Thrombocytopenia causes few signs or symptoms with platelet counts in the range of 50–100 × 109/L that represent moderate-to-severe thrombocytopenia. Diseases such as dengue, idiopathic thrombocytopenic purpura, hypersplenism, aplastic anaemia, chikungunya, and drug-induced thrombocytopenia result in a low thrombocyte count in blood (Aster and Bougie Carica papaya Linn. (Caricaceae) leaf has been used in folk medicine since centuries for wound healing and in treatment of blood disorders, jaundice, and malaria , flavonoids , cyanogenic glycosides, and glucosinolates , β-carotene (97%), gallic acid and rutin (94%) were procured from Sigma-Aldrich . The HPTLC plates Si 60 F254 (10 × 20 cm) were purchased from E. Merck . The myricetin (98%), trans-ferulic acid (99%), caffeic acid (98%), and kaempferol (98%) were purchased from Chromadex . Solvents used for chromatography were of HPLC grade, and distilled water was obtained by a Milli-Q plus water purification system . Edoxin (cyclophosphamide injection) was procured from Apollo Pharmacy.Formic acid, acetonitrile, methanol, and anhydrous sodium sulphate were purchased from J.T. Baker , while diethyl ether, methyl C. papaya were collected in July 2012 from Jamia Hamdard Campus. The leaves were authenticated from NISCAIR by taxonomist Dr. H. B. Singh (NISCAIR/RHMD/Consult/-2012-13/2158/164).The tender leaves of The fresh leaves were cleaned of the debris matter, depetiolated after removing woody stalks, and washed with purified water. The cleaned leaves (300 g) were manually chopped and crushed directly for one min in a grinder with the addition of small amount of distilled water. The volume of mixture obtained was made up to 1000 mL with the addition of distilled water and kept at ambient temperature for 24 h with occasional shaking. It was filtered off using muslin cloth and the marc left was washed with distilled water. The filtrate and washings were pooled together and the final volume was made up to 1000 mL. The collected juice/extract was subjected to freeze drying and stored at −20 °C until further use. The freeze-dried powder was subjected to quantitative estimation of phytoconstituents for standardization and quality control prior to use for biological activity.trans-ferulic acid, and kaempferol. Chromatography was performed on 20 × 10 cm aluminium HPTLC plates coated with 0.2 μm layers of silica gel 60F254. Samples were applied as bands of 4.0 mm wide, 8.3 mm apart by the use of a CAMAG (Switzerland) Linomat V sample applicator fitted with a microliter syringe. A constant application rate of 120 nL/s was used. Linear ascending development, to a distance of 80 mm, with toluene:ethyl acetate:formic acid  as mobile phase, was performed in a 20 × 10 cm twin-trough glass chamber, saturated with mobile phase for 15 min. The developed plates were air-dried and scanned at 320 nm by CAMAG TLC densitometric scanner III operated by winCATS software. The source of radiation was a tungsten lamp, the slit dimensions were kept at 4.0 × 0.3 mm, and the scanning speed was 10 mm/s. The method was validated as per the ICH guidelines for calibration, linearity, precision, accuracy, robustness, specificity, LOD, and LOQ as per previously described methods  equipped with a binary solvent delivery system, an auto sampler, column manager, and a tunable MS detector . Chromatographic separation was performed on a Water’s ACQUITY UPLCTM BEH C18 (100.0 × 2.1 mm ×1.7 μm) column at 40 ± 5 °C. The mobile phase used for UPLC analysis consisted of 0.5% formic acid (A) and acetonitrile (B), at a pH of 2.7, which was degassed before analysis, and the gradient elution was 2 to 100% (B) in 40 min at a flow rate of 0.5 mL/min and 25 °C. The injection volume was 20 μL. The column and auto sampler were maintained at 40 ± 5 °C and 25 ± 5 °C, respectively, and the pressure of the system was set to 15000 psi.The LC-MS was performed on Water’s ACQUITY UPLCTM, Micromass MS Technologies, Manchester, UK). The nebulizer gas was set to 500 L/h, the cone gas was set to 50 L/h, and the source temperature was set to 100 °C. The capillary voltage was set to 3.0 kV and the sample cone voltage was set to 40 kV. Argon was employed as the collision gas at a pressure of 5.3 × 10−5 Torr. The Q-TOF PremierTM was operated in V mode with resolution over 8500 mass with 1.0 min scan time and 0.02 sec interscan delay. The accurate mass and composition for the precursor ions and for the fragment ions were calculated using MassLynx v 4.1 software incorporated with the instrument.The mass spectrometry was performed on a quadruple orthogonal acceleration time of flight tandem mass spectrometer  were procured from Central Animal House Facility, Jamia Hamdard, New Delhi. The animals were divided into four groups of six each. Group I and group II served as normal and toxic controls, respectively. Groups II–IV received CP  for first three days, consecutively. Groups III and IV served as treatment groups receiving, SCPLE, 50 and 150 mg/kg b.wt, p.o., respectively, from day 1 to day 14. In the absence of any treatment, both groups I and II received 0.8 mL normal saline, orally. First treatment with CP was counted as day one. One hour after last treatment, blood samples were collected from each animal and subjected to Coulter Counter for analyses of platelet count (PC), total leucocyte count (TLC), differential leucocyte count (DLC) including neutrophils, lymphocyte, monocyte, eosinophil, basophil, bleeding time, and clotting time Duke . The dos8 cells of fresh sheep red blood cells (SRBC) suspension, i.p, on 6th day for humoral antibody and delayed-type hypersensitivity response.Fifty-four female Swiss albino mice, weighing 30–45 g, procured from Central Animal House, Jamia Hamdard, New Delhi. The animals were divided into nine groups of six each and maintained as per the standard protocol of CPCSEA guidelines. Group I served as normal control received normal saline (0.2 mL), groups II–IX received CP  consecutively for three days on 1st, 2nd, and 3rd day of experiment, and groups IV–IX received CP  on day 8, 9, and 10. Groups II, IV, VI, and VII served as toxic control, whereas groups III, V, VII, and IX served as treatment and received SCPLE (150 mg/kg/day p.o.). The animals of groups II and III were immunized by injecting 0.1 mL of 1 × 108 SRBC in the left hind footpad and the right footpad with the same volume (0.1 mL) of normal saline. The thickness of the footpad was measured using digital vernier caliper after SRBC administration at 0, 24, and 48 h. The pre- and postchallenge difference in the thickness of footpad was expressed in mm and taken as a measure of delayed-type hypersensitivity , instead of 50 mg/kg i.p. The blood was collected for TLC prior to and after 3rd day of injection of neutropenic dose of CP  on 10th day of study. The blood was collected at 2-min and 10-min intervals after injection of ink dispersion. The sodium carbonate solution  was added to the blood samples to lyse the erythrocytes. Absorbance was taken at 675 nm using spectrophotometer. The mean of phagocytic index was calculated for each group of animals .The values were expressed as mean ± SEM. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Student’s The FDP yielded 4.843% w/w of fresh juice, which was used for HPTLC analysis and UPLC-qTOF/MS fingerprinting for quality control as well as for determination of antioxidant, antithrombocytopenic, and immunomodulator activity.trans-ferulic acid, and kaempferol at Rf value 0.39 ± 0.01, 0.44 ± 0.02, 0.50 ± 0.01, and 0.55 ± 0.01, respectively  in CPL mother extract. The interday, intraday, and interlaboratory precision at three concentration levels , in terms of %RSD, was in the range of 0.05–2.00 for all the compounds. The low values of %RSD obtained after incorporating small but thoughtful changes in wavelength ; (ii) robustness of the HPTLC method for estimation of mixed standards by changing detecting wavelengths (n = 3) showing Levy-Jennings Plot of %RSD for markers; (B(i)) robustness of the HPTLC method for estimation of mixed standards by changing detection of mobile phase composition (n = 3); (ii) robustness of the HPTLC method for estimation of mixed standards by changing detecting of mobile phase composition (n = 3) showing Levy-Jennings plot of %RSD for markers.(A(i)) Robustness of the HPTLC method for estimation of mixed standards by changing detecting wavelengths  in ESIMassLynx . The retMassLynx  shows thMassLynx .50 values of the butylated hydroxy anisole, butylated hydroxy toluene, ascorbic acid, and SCPLE were 2.03 ± 0.19, 0.95 ± 0.30, 4.17 ± 0.02, and 4.93 ± 0.98 μg/mL, respectively. The β-carotene content in 100 g of SCPLE was found to be 779.69 ± 5.55 μg.The metabolite analysis data showed that total flavonoid content was found to be 16.74 ± 0.50 QE/g in SCPLE, whereas total phenolic content was 29.54 ± 0.02 mg GAE/g. CPL exhibited dose-dependent inhibition of DPPH radical in a stoichiometric manner. The IC3 cells/mm3, TLC 7.88 × 103 cells/mm3, and neutrophils 68%, while after administration of SCPLE on day 15, the PC was 1014.83 × 103 cells/mm3, TLC 12.90 × 103 cells/mm3, and neutrophils 85% (p < 0.01).The present study examined the platelet increasing effect on oral administration of SCPLE on oral administration in both normal and thrombocytopenic rats. It was observed that PC, TLC, and neutrophils decreased from normal level. On day 3rd, PC was 408.5 × 10hils 85% . The SCPp < 0.05) lowered after oral administration of SCPLE in groups III and IV, as compared to toxic control  (p < 0.01) in extract-treated groups, when compared with toxicant group receiving CP  . Signifiiving CP .p < 0.01). Co-administration of SCPLE produced reversal of TLC in treatment groups. The neutrophil count (%) was highly reduced in CP-treated toxic control, when compared to initial values on day ‘0’. The CP-induced neutropenia model is dependent on the effect of drugs on haematopoietic system. The SCPLE also showed good carbon clearance test and high phagocytic index (p < 0.001). The SCPLE has significantly inhibited the production of TNF-α in CP-treated animals as shown in The neutropenic dose  of CP significantly reduced the TLC in control animals  at Rf 0.39 ± 0.01, 0.44 ± 0.02, 0.50 ± 0.01, and 0.55 ± 0.01, respectively. The CPL aqueous extract showed the presence of myricetin, trans-ferulic acid, caffeic acid, and kaempferol using HPTLC. Comparison of the UV spectral characteristics of all peaks for the standards revealed the identity of same peaks in the sample. The calibration curves were linear in the range of 25–1250 ng for myricetin, trans-ferulic acid, caffeic acid, and kaempferol. The trans-ferulic acid was present in higher amount (1110.86 ± 2.97 ng/g) of all the active ingredients that were recorded in CPL aqueous extract, which are in corroboration with earlier report , being used as immunomodulator in the indigenous system, have been reported to modulate the immune system in various The increase in the carbon clearance index reflects the enhancement of the phagocytic function of mononuclear macrophage and nonspecific immunity by the cells of reticuloendothelial system (RES). The RES is a diffuse system consisting of phagocytic cells, which play an important role in the clearance of particles from blood stream. When colloidal carbon particles in the form of ink are injected directly into the systemic circulation, the rate of clearance of carbon from blood by macrophage is governed by an exponential equation (Sharma and Rangari The results of present investigation clearly indicate that SCPLE produces increase in the PC and fall in the various biochemical parameters namely bleeding time and clotting time because of the formation of platelet plug at the time of excessive bleeding, whereas increase in TLC and DLC associated with increased thrombocytes without possessing toxic effect on any organ. Hence, SCPLE can be used for a longer time for diseased conditions, where PC drastically decreases and it is also used to boost the body’s immunity.in vivo studies proved usefulness of SCPLE in thrombocytopenia, which was evidently confirmed by histological studies of spleen and liver followed by assessment of immunomodulatory activity. The oral administration of 150 mg/kg body weight of SCPLE caused significant increase in PC, TLC, and DLC with reduced bleeding time and clotting time without any acute toxicity. The proposed study therefore provides a great future of SCPLE for dengue fever-related thrombocytopenia in the era of phytopharmaceuticals.The developed HPTLC was used for simultaneous quantitative analysis of phenolic acids and flavonoids in SCPLE. UPLC-qTOF/MS method was developed for qualitative analysis of six phenolics, two alkaloids, sixteen hydroxycinnamic acid derivatives, and flavonoids to develop metabolite signature of SCPLE. The results of


A liquid chromatography-tandem mass spectrometry method had been developed and fully validated for simultaneous determination of paclitaxel, docetaxel, vinblastine, vinorelbine, pemetrexed, carboplatin, etoposide, cyclophosphamide, ifosfamide, gemcitabine, irinotecan, and SN-38 (an active metabolite of irinotecan) in human plasma from cancer patients after intravenous drip of chemotherapy drugs. One-step solid-phase extraction was successfully applied using an Ostro sample preparation 96-well plate for plasma samples pretreated with acetonitrile containing 0.1% formic acid. Chromatographic separation was achieved on an Atlantis T3-C18 column  with gradient elution using a mobile phase consisting of acetonitrile and 10 mM ammonium acetate plus 0.1% formic acid in water, and the flow rate was 0.25 mL/min. The Agilent G6410A triple quadrupole liquid chromatography-mass spectrometry system was operated under the multiple reaction monitoring mode with an electrospray ionization in the positive mode. Linear range was 25.0–2500.0 ng for paclitaxel, 10.0–1000.0 ng for docetaxel and SN-38, 100.0–10000.0 ng for vinorelbine and pemetrexed, 10.0–10000.0 ng for vinblastine and irinotecan, 1.0–1000.0 ng for cyclophosphamide and ifosfamide, 50.0–5000.0 ng for carboplatin, etoposide, and gemcitabine. Linearity coefficients of correlation were >0.99 for all analytes. The intraday and interday accuracy and precision of the method were within ±15.0% and less than 15%. The mean recovery and matrix effect as well as stability of all the analytes ranged from 56.2% to 98.9% and 85.2% to 101.3% as well as within ±15.0%. This robust and efficient method was successfully applied to implement therapeutic drug monitoring for cancer patients in clinical application.Therapeutic drug monitoring for anticancer drugs could timely reflect  Cancer was gradually becoming an heavy burden for both developed and developing countries, and its incidence and mortality had increased awfully in recent years along with the aging of people and the deterioration of environment as well as the changing of lifestyles, for example, smoking, alcohol abuse, and massive intake of red meat. There were about 14.1 million new cancer cases and 8.2 million deaths in the worldwide in 2012 according to the GLOBOCAN report [In clinical application, the ways for treatment of cancer often included radiotherapy, chemotherapy, and surgery. Among that, chemotherapy was a potent and adjuvant method served for the surgery and sporadic cancer cells eradication. In recent years, enormous progress had been made in chemotherapy agents, and drug combinations containing multiple cytotoxic anticancer agents or biotherapy occupied the dominant status and yielded a good paradigm for cancer treatment , 3. DrugThe area under the concentration-time curve, steady-state concentration, and concentration over the threshold had a close relationship with the therapeutic results, and they had been considered as potent biomarkers for the individualized treatment in recent years . ConcentA HPLC-MS/MS method for simultaneous determination of seven anticancer agents had been developed and reported in our laboratory, but two steps of extraction hindered its high throughput in clinical application . Thus, tμm) was self-made in the laboratory by a Milli-Q reagent water system  and was used throughout. Waters Ostro 96-well plate (25 mg) was used for sample pretreatment . Human blank plasma was donated by Shanghai Red Cross Blood Center .Paclitaxel, docetaxel, vinblastine, vinorelbine, pemetrexed, carboplatin, etoposide, cyclophosphamide, ifosfamide, gemcitabine, irinotecan, SN-38, and vindoline  were purchased from Sigma-Aldrich Corporation . All were corrected for purity and salt forms when weighed or diluted for standard stocks, whose chemical structures are shown in All experiments were carried out on an Agilent 1200 series HPLC system including an online degasser, a quatpump, an autosampler, and a column oven and interfaced to an Agilent 6410A triple-quadrupole mass spectrometer with an electrospray ionization source . All data were acquired and analyzed using Agilent Masshunter data processing software .18 analytical column . The column was equilibrated and eluted with a mixed mobile phase consisting of acetonitrile and water containing 0.1% formic acid plus 10 mM ammonium acetate at a flow rate of 0.25 mL/min. The mobile phase was degassed automatically using the online degasser system. Mobile phase A was water containing 0.1% formic acid plus 10 mM ammonium acetate. Mobile phase B was acetonitrile. The gradient variation started with 100% A and maintained for 1 min, then switched to 100% mobile phase B at 1.01 min and lasted until 9 min, and finally switched back to 100% mobile phase A at 9.01 and lasted until 10 min, after which the system was returned to the initial condition. Under these conditions, the analytes coeluted with the IS within 9 min. The column temperature was maintained at 35°C. The injection volume was 10 μL, and the analysis time was 10.0 min.The chromatographic separation was achieved on an Atlantis T3-CThe mass detection was achieved using electrospray ionization in the positive mode with the spray voltage set at 4000 V. Nitrogen was used as nebulizer gas, and nebulizer pressure was set at 40 psi with a source temperature of 105°C. Drying gas (nitrogen) was heated to 350°C and delivered at a flow rate of 10 L/min. High-purity nitrogen was used as collision gas at a pressure of about 0.2 MPa. Quantitation detection was performed in the multiple reaction monitoring (MRM) mode. The peak widths of precursors and product ions were maintained at 0.7 amu at half-height in the MRM mode.μg/mL for paclitaxel, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, and 10.0 μg/mL for docetaxel and SN-38, 1.0, 2.0, 5.0, 1.0, 25.0, 50.0, and 10.0 μg/mL for vinorelbine and pemetrexed, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 μg/mL for vinblastine and irinotecan, 10.0, 50.0, 100.0, 500.0, 1000.0, 5000.0, and 10000.0 ng/mL for cyclophosphamide and ifosfamide, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, and 50.0 ng/mL for carboplatin, etoposide, and gemcitabine. Calibration standards were prepared by 10 times dilution of the corresponding combined working solutions with blank human plasma to obtain final concentrations in the range of 25.0–2500 ng/mL for paclitaxel, 10.0–1000.0 ng/mL for docetaxel and SN-38, 0.1–10.0 μg/mL for vinorelbine and pemetrexed, 10.0–10000.0 ng/mL for vinblastine and irinotecan, 1.0–1000.0 ng/mL for cyclophosphamide and ifosfamide, and 50.0–5000.0 ng/mL for carboplatin, etoposide, and gemcitabine. Quality control (QC) samples were also prepared in the same way . The QC samples were stored at −20°C and brought to room temperature (25°C) for thawing before being processed.The stock solutions of paclitaxel, docetaxel, vinblastine, vinorelbine, etoposide, cyclophosphamide, ifosfamide, and vindoline (IS) were individually prepared in methanol, the stock solutions of pemetrexed, carboplatin, and gemcitabine were individually prepared in water, and the stock solutions of irinotecan and SN-38 were prepared in DMSO to obtain final concentrations at 1.0 mg/mL. All stock solutions were stored at −20°C. The stock solution of each analyte was further diluted with 10% methanol (V : V) to obtain a series of work solutions at concentrations of 0.25, 0.5, 1.25, 2.500, 6.25, 12.5, and 25.0 μL and 1000 μL pipetting tools  were utilized for liquid transfer steps. A 100 μL aliquot of samples was added to the designated well followed by adding 20 μL of IS working solution (100 ng/mL). A 300 μL aliquot of acetonitrile containing 1% formic acid was added to each well to precipitate the plasma proteins. All the wells were vortex mixed by aspirating 3 times with pipette. The mixtures were pulled through the cartridges to deprive the phospholipids using 96-well positive pressure processor  under 60 psi for 5 min. A 1 mL collection plate was used for collecting the eluents. After transferring to Eppendorf tubes, the content was evaporated under a gentle nitrogen stream at 45°C. A 100 μL of the initial mobile phase was added to the tube, and the residual was reconstituted after vortex mixing for 3 min and then centrifuged for 10 min at 2500 ×g. The supernatant was transferred to a 1.5 mL glass autosampler vial with inserts, and 10 μL of the supernatant was injected into the HPLC-MS/MS system for analysis.Sample pretreatment was performed on Ostro™ 96-well plate to remove phospholipids and proteins . An eight-channel 100 2), vinblastine (10 mg/m2), vinorelbine (25 mg/m2), and cyclophosphamide (1000 mg/m2) were obtained at 0 h and 24 h after intravenous infusion. Other samples containing ifosfamide, etoposide, gemcitabine, carboplatin, and pemetrexed were collected at 0 h, 3 h, and 24 h after intravenous infusion at dosages of 1200 mg/m2, 80 mg/m2, 2200 mg/m2, 330 mg/m2, and 800 mg/m2, respectively. For paclitaxel, venous blood samples were harvested at 0, 6, 24, and 48 h after intravenous infusion at dosages of 210 mg/m2, 240 mg/m2, or 270 mg/m2, while for docetaxel at dosages of 100 or 120 mg/m2 samples were collected in the same points as paclitaxel. Plasma samples were immediately separated by centrifugation at 2000 ×g and 4°C for 10 min. Plasma samples were transferred to 1.5 mL Eppendorf tubes and stored at −80°C until analysis. All samples were processed within 1 h.The research protocol was reviewed and approved by the Ethical Committee of Changzheng Hospital  and carried out in Changzheng Hospital. Informed consent was signed by all the recruited patients. Human venous blood samples were collected in heparinized vacuum tubes and gently placed in an ice bath until centrifugation. Samples for irinotecan .For specificity, comparison of responses in spiked and blank samples from at least 6 lots was performed. The responses of interferences not more than 20% for analytes and 5% for IS were acceptable.Matrix effect and recovery were assessed in three replicates at three concentration levels  for paclitaxel , docetaxel and SN-38 , vinorelbine and pemetrexed , vinblastine and irinotecan , cyclophosphamide and ifosfamide , and carboplatin, etoposide, and gemcitabine . The matrix effect was the ratio of peak area in the spiked postextraction samples to the concentration corresponding solvent substituted samples, and the recovery was the ratio of peak area in the spiked samples concentration corresponding spiked postextraction samples. The IS was also assessed in the same way at the concentration of 100 ng/mL.Interday and intraday precision and accuracy were also assessed in five replicates at three concentration levels . Samples were analyzed in three analytical lots in separate days (at least 2 days), and the RSD% for interday and intraday precision not more than 15% was rational. For intraday and interday accuracy, RE% (relative error) within 15% was considered acceptable.χ2 weighted linear least-squares regression model. The LLOQ was set at the lowest point of the calibration curve, which must possess a 10 times response higher than interference, and the deviation for the precision and accuracy should not be more than 20% and should be within ±20%.Linearity of each analyte was evaluated in three analytical lots, and calibration curves were regressed from IS-adjusted peak area versus corresponding concentrations in at least six calibration standards using a 1/Stability including long-term stability (3 months), short-term stability (24 h in an autosampler), and three frozen-thaw cycle stability was evaluated using QC samples at three levels . The calibration curve for each analyte was employed to obtain the measuring concentrations, and the deviation from nominal concentration within 15% was conformed to the criterion.18 and C8 columns, such as Zorbax SB-C18, Zorbax SB-C8, Xselect/Xbridge-C18, C8, and Atlantis T3-C18, were tested. Elution of pemetrexed, carboplatin, and gemcitabine needed high percentage (at least 80%) of organic solvent in the mobile phase; however, high proportion of organic solvent (85%) caused that the analytes could not be totally separated from the endogenous interfering materials on the SB-C18 and C8 column. Finally, the Atlantis T3-C18 column provided sufficient retention and suitable separation medium for twelve analytes based on its stronger retention ability. Several mobile phases had been tested: 0.05% formic acid, 0.1% formic acid, 0.05% acetic acid, 0.1% acetic acid, 5 mmol/L ammonium acetate, 10 mmol/L ammonium acetate/water solutions in combination with either methanol or acetonitrile. With methanol as organic solvent, moderate peak tailing was observed, while with acetonitrile, split peaks were shown. As the temperature rises up, the resolution improved with a narrower peak width. In conclusion, the most appropriate mobile phase was acetonitrile and 10 mmol/L ammonium acetate plus 0.1% formic acid in water at a flow rate of 0.25 mL/min, and the column temperature was kept at 30°C. The twelve analytes and the IS were at first characterized by full-scan mode and then product ions mode to ascertain their precursor ions and product ions which were utilized for constructing the MRM mode. The full-scan mode showed that the ionization of the analytes was more suitable in the positive mode. The parameters for fragmentor energy and collision energy were optimized and listed in The chromatographic conditions, especially the composition of the mobile phase and types of columns, were optimized through several tests to achieve good resolution and symmetric peak shapes for analytes and the IS, as well as shorter run time. A number of Ctert-butyl methyl ether, or mixed solvent for LLE) [Due to the complex composition of plasma, a sample pretreatment was often needed to remove protein and other potential interfering materials prior to LC-MS/MS analysis. Liquid-liquid extraction (LLE) with different organic solvents and protein precipitation (PPT) with acetonitrile or methanol were evaluated as sample pretreatment techniques in our earlier studies. Initially, several conventional PPT  and LLE for LLE) –29, but Comparison of blank and spiked human plasma chromatograms  indicateMatrix effect commonly occur due to interfering materials that coexist and coelute in the LC system in the biofluid; generally speaking, the interfering materials that were eluted together with the analytes could influence mostly the response of analytes. The PPT, which usually utilized organic solvent  to strip the proteins in the biofluid by destroying the structure of them, had the lowest ability to eliminate the coexisting interfering materials. Lipids, fats, and other low molecular weight substances could not be striped by the PPT. Part selective extraction of analytes could be completed by an LLE and SPE pretreatment method, but the operation and solvent selection of the two extraction method was relatively complicated. In this study, one-step extraction by an Ostro sample preparation 96-well plate was successfully developed to obtain a high recovery and low matrix effect. The operation simply consisted of PPT and filtration and could be accomplished within 5 min, which promised a high-throughput sample pretreatment. The results showed that a matrix effect ranged from 85.2% to 101.3% and recovery ranged from 56.2% to 98.9%. The matrix effect and recovery were stable and conformed to the pharmacopeia criterion. All data are summarized in χ2 weighing factor. The squares of the linear correlation coefficients were all over 0.99. Typical regression equations for the calibration curves were summarized in S/N) >10 at the LLOQ was observed for all the analytes. These LLOQs were sufficient for clinical drug monitoring of the eleven commonly used anticancer drugs and one active metabolite.Calibration curves were constructed by plotting the peak area ratios  of calibration standards versus nominal concentrations from spiked samples. The regression model was constructed based on linear regression with or without intercepts and weighing factors. The best linearity and least-squares residuals for the calibration curves were achieved with a 1/Three levels of QC samples  were chosen to analyze the interday and intraday precision and accuracy. The results showed a good precision and accuracy with intraday and interday precision less than 15% and accuracy within ±15%. The stability of analytes in long-term cryopreservation, an autosampler, and three frozen-thaw cycles was investigated. The analytes were found to be stable in human plasma stored for 3 months at −20°C and in an autosampler at room temperature for 24 h (<5% reduction). After three freeze-thaw cycles, no obvious reductions (less than 15%) were observed for all the analytes .Totally, 132 samples (69 from women and 63 from men) from 48 lung cancer patients were collected. The means (ranges) of age and body weight were 52.5 (18–82) years and 62.0 (51–72) kg, respectively. This entirely validated method was successfully applied for the determination of eleven commonly used anticancer drugs and one active metabolite concentration in the human plasma after administration. The results showed a big difference in patients administered with the same drugs in the same dosage, and the linearity range of this method covered all the concentration variations from different sampling points, which proved an extensive applicability of this method. All data are shown in A simple, efficient, and sensitive HPLC-MS/MS method was successfully developed and was suitable for simultaneous determination of eleven commonly used anticancer drugs and one active metabolite in the human plasma from cancer patients. The analytical time was 10 min, and the LLOQ for all analytes was not more than 100 ng/mL. Owing to the application of an Ostro 96-well plate and a special column, this method achieved a simple and efficient sample preparation and separation process, which paved the way for the high throughput and extensive scope in clinical application for routine TDM of anticancer drugs to gain real-time drug exposure so that individualized therapeutic regimen may be promoted.


Securidaca longipedunculata Fresen is an indigenous medicinal plant in Africa that has an important place in both traditional and modern medicine. This plant is endangered because of high seed dormancy, low germination rate, and over exploitation. Therefore, micropropagation method is important to address these problems. The objective of this study is to develop a micropropagation protocol for S. longipedunculata from shoot tip explants.Among different Clorox concentrations, seeds sterilized with 10% Clorox for 10 min resulted in 85% decontamination and 80% germination. Among different media used to evaluate the rate of seed germination, seeds that were de-coated and transversally cut at the tip and cultured on basal MS medium resulted in 100% germination. The highest percentage of shoot initiation (87%) was obtained on MS medium containing 1.0 mg/l 6-Benzylaminopurine (BAP). The highest mean shoot number per explant (8.5 ± 0.69) was achieved on MS multiplication medium containing 1.5 mg/l BAP in combination with 0.1 mg/l Indole-3-butyric acid (IBA). The highest mean number of roots per explant (3.73 ± 0.69) was obtained on MS medium containing 2.0 mg/l Indole-3-acetic-acid (IAA). Among plantlets transferred to greenhouse, 60% survived after acclimatization.S. longipedunculata that contributes to its conservation and genetic improvement.This micropropagation protocol can be used for mass propagation of  Securidaca longipedunculata Fresen is a small tree belonging to family Polygalaceae. It grows up to 12 m tall, with an often flattened or slightly fluted bole. It is an indigenous medicinal plant in Africa and plays an important role in both traditional and modern medicine. This tree is also commonly used as a pesticide against beetles in stored grains [It is distributed in a wide range of climates ranging from subtropical, hot, and arid climate to summer rainfall and equatorial humid. It grows in different vegetation ranging from semi-arid scrub to dense forest, including bush habitats and gallery forests and woodland . It is sThe root and bark are taken orally either powdered or as infusion for treating malaria, stomach problems, toothache, headache, sleeping sickness, cough, chest complaints, snakebite, and wound dressing and roots are used to kill seed storage pests. An infusion of the roots is used as a mouthwash in cases of toothache and is applied to cuts on the legs to treat inflammation. Powder from the burned roots is rubbed into small incisions made on the forehead to relieve headaches. Seeds are used for headache, fever, and rheumatism; leaves for snakebite, venereal diseases, and coughs, whereas the bark is used for stomach problems and as an arrow poison antidote .S. longipedunculata has different chemical constituents including methylsalicylate, flavonoids, alkaloids elymoclavine, and dehydroelymoclavine, an ergoline compound and cinnamonic acid and the xanthones: 1,7-Dimethoxy-2-hydroxy-xanthone and 1, 4-dihydroxy-7-methoxy-xanthone [xanthone . A numbexanthone .S. longipedunculata has been recognized by the findings of different bioactive metabolites isolated from the bark yielding such as oleanolic acid, glycoside and alkaloid securiene used in treating convulsion in children, increased blood pressure and paralysis following infectious disease [S. longipedunculata induces apoptosis in brain tumor confirming its folkloric use in cancer management [The medicinal importance of  disease . It is u disease . Recent nagement .Plasmodium falciparum [The alkaloid securinine confers activity against lciparum . Some fllciparum , 12.S. longipedunculata is threatened because of the fact that roots are the target for people using this plant, which makes it difficult for the plant to survive constant harvesting. It suffers from uncontrolled harvesting for use in local medicines, as well as periodic droughts. It is considered vulnerable due to increasing threats to their habitats.As the fruits usually stay on the tree for a year or more, it has been suggested that seeds should not be sown until it is 1-year old, but the seeds seem to lose viability quickly and germination is erratic. It is best to sow a seed soaked in cold water for 24 h in the soil and cover with grass and water until raining. It is difficult to cultivate largely because germination is poor and even after germination, seedling growth is slow and planting out is difficult because the taproot is broken easily . S. longS. longipedunculata in agro-forestry systems. In vitro propagation of S. longipedunculata has considerable benefits for its availability as planting material and germplasm conservation. The protocol can facilitate mass propagation of superior genotypes for wider planting in agro-forestry systems or in its natural habitat where restoration and conservation is envisaged. This enables wise utilization of the plant through cultivation of elite plants for medicinal purposes in large quantities sustainably. In the present study, different germination rates of both coated and de-coated seeds of S. longepedunculata under different conditions were investigated, and in vitro propagation protocol was developed.Little effort has also been made to develop propagation methods to facilitate planting of Matured seeds were collected from Metema (Gendawuha), near the border with Sudan, about 1,000 km west of Addis Ababa, Amhara regional state, Ethiopia. The plant material was identified by the first author. Moreover, a voucher specimen of this material had been previously deposited in a publicly available National Herbarium. Seeds were washed under running tap water using detergent, washed in 70% ethanol for 2 min and rinsed three times with sterile double-distilled water followed by sterilization in 3%, 5%, 10%, and 15% sodium hypochlorite (Clorox)  containing three drops of Tween-20  for 10 min. The seeds were then washed three times with sterile double-distilled water and cultured on basal MS  medium (Seeds were germinated in four conditions. 1) Both coated  for 30 s and rinsed three times with sterile double-distilled water followed by sterilization in 10% Clorox containing three drops of Tween-20 for 10 and 15 min. After sterilization, the explants were washed three times with sterile double-distilled water.−2 s−1 and 16 h photoperiod provided by cool-white fluorescent lamp . For each treatment, a total of 30 explants were used. There were six explants per jar with five replications. The experiment was repeated once. Number of dead and initiated shoots was recorded.The shoots were trimmed to about 1.0 cm and cultured in baby food jars each containing 30 ml shoot initiation medium. The shoot initiation medium was MS medium containing 6-Benzylaminopurine (BAP)   in combination with 0.5 mg/l thidiazuron (TDZ) or TDZ alone  , 30 g/l sucrose, 0.7% (w/v) agar . The pH of the medium was adjusted to 5.8 before addition of agar. Basal MS medium was used as control. The cultures were maintained at a temperature of 25 ± 2 °C under light intensity of 20 μmol mInitiated shoots were cultured in Magenta GA-7 culture vessels  containing 50-ml shoot multiplication medium. Shoot multiplication medium was MS medium containing different concentrations of BAP  in combination with Indole-3-butyric acid (IBA) , TDZ  in combination with IBA , or TDZ  in combination with α-Naphthalene acetic acid (NAA) . Basal MS medium was used as control. The cultures were maintained at the same condition as for shoot initiation. For each treatment, a total of 30 explants were used. Number of shoots and shoot length were recorded after 4 weeks of culture. The whole experiment was repeated once.Rooting was done using full- and half-strength MS medium containing IBA , Indole-3-acetic-acid (IAA) , or NAA . Basal MS medium was used as control. Shoots were maintained for a week in the dark and then transferred to the same condition as for shoot initiation for 3 weeks. Number of shoots that produced roots, number of roots per shoot, and length of roots were recorded after 5 weeks of culture.After 5 weeks in rooting medium, rooted shoots were removed from culture vessels and the roots were washed thoroughly under tap water and transferred to pots containing a mixture of soil, compost, and sand in a ratio of 2:1:1, respectively. Each pot was covered with a polyethylene bag and kept in a laboratory for 1 week before being transferred to glasshouse. The polyethylene bags were removed after a week of transferring to glasshouse. The plants were watered every day. The number of survived plants in the glasshouse was recorded after a month.p ≤ 0.05 was considered significant. Data were subjected to analysis of variance and variables that showed significant difference were compared by the LSD at 5% probability.Statistical analysis of quantitative data was carried out by SPSS computer software of version 20. A difference at probability level of The highest percentage of decontaminated seeds (90%) was obtained at 15% Clorox, but the germination percentage was reduced to 20% (Table p ≤ 0.05). However, no significant difference was observed among 5%, 10%, and 15% Clorox concentrations with respect to seed decontamination, but the increasing concentration of Clorox to 15% negatively affected seed germination.ANOVA result showed that Clorox concentrations significantly affected percentage of decontaminated seeds and germinated seeds  of seeds was observed within 7–10 days on basal MS medium, when the seeds were de-coated and transversally cut at the tip. As shown in Table 00% of seThe use of a semi-solid MS medium containing BAP or in combination with TDZ or TDZ alone resulted in shoot initiation within a week. The highest percentage (87%) of shoot initiation was obtained on MS medium containing 1.0 mg/l BAP followed by 67% of shoot initiation on 1.0 mg/l BAP in combination with 0.5 mg/l TDZ Table . The micShoot number was highly influenced by concentration and type of the growth regulators. Different combinations of BAP, IBA, TDZ, and NAA resulted in different responses. Different concentrations of BAP in combination with IBA produced better shoot number and length per explant than other growth regulator combinations Figs. d and e. In terms of shoot length, the highest mean shoot length 2.11 ± 0.45 cm) was obtained on medium containing 2.5 mg/l BAP in combination with 0.1 mg/l IBA. However, there was no significant difference between shoots that were produced on 2.5 mg/l BAP in combination with 0.1 mg/l IBA and 2.5 mg/l BAP in combination with 0.01 mg/l IBA. The lowest mean shoot length (0.96 ± 0.40 cm) was recorded on MS medium containing 0.5 mg/l BAP in combination with 0.5 mg/l IBA  was produced on MS medium containing 0.5 mg/l TDZ alone followed by shoots cultured on MS medium containing 2.5 mg/l TDZ in combination with 0.5 mg/l IBA, which was 5.23 ± 0.49. The lowest mean shoot number (2.10 ± 0.12) was recorded on basal MS medium (Table The highest mean shoot length (1.55 ± 0.09 cm) was obtained on MS medium containing 0.5 mg/l TDZ while the lowest mean shoot length (0.31 ± 0.02 cm) was produced on MS medium containing 1.0 mg/l TDZ in combination with 0.5 mg/l IBA.In case of TDZ and NAA combinations, the highest mean shoot number per explant (4.70 ± 0.32) was obtained on MS medium containing 1.5 mg/l TDZ in combination with 0.5 mg/l NAA followed by MS medium containing 1.5 mg/l TDZ in combination with 0.1 mg/l NAA, which produced 4.56 ± 0.24 shoots per explant. However, there was no significant difference between mean shoot numbers per explant obtained on medium containing 1.5 mg/l TDZ in combination with 0.1 mg/l NAA and 0.5 mg/l TDZ in combination with 0.01 mg/l NAA. The lowest mean shoot number per explant (2.10 ± 0.12) was recorded on basal MS medium (Table TDZ in combination with NAA produced relatively shorter shoots. The highest mean shoot length (1.22 ± 0.03 cm) was obtained on growth regulator-free MS medium while the shortest mean shoot length (0.16 ± 0.01 cm) was produced on MS medium containing 2.5 mg/l TDZ in combination with 0.01 mg/l NAA  was obtained on MS medium containing 1.5 mg/l BAP in combination with 0.1 mg/l IBA. On the other hand, the lowest mean shoot number per explant (2.10 ± 0.12) was obtained on basal MS medium. MS medium containing 2.5 mg/l BAP in combination with 0.1 mg/l IBA resulted in the highest mean shoot length (2.11 ± 0.45 cm) while the shortest mean shoot length (0.16 ± 0.01 cm) was obtained on MS medium containing 2.5 mg/l TDZ in combination with 0.01 mg/l NAA.Rooting was observed 4 weeks after culture of the shoots on root induction medium. However, most of the shoots developed roots by 5 weeks Fig. f. The hiThe highest mean root length (0.34 ± 0.02 cm) was observed on full-strength MS medium containing 3.0 mg/l IAA. However, no significant difference was observed among roots developed on full-strength MS medium containing 3.0 mg/l IAA and half-strength MS medium containing 0.5 mg/l IAA. Among all concentrations, the lowest mean root length (0.07 ± 0.01 cm) was observed on half-strength MS medium containing 0.5 mg/l NAA or 0.5 mg/l IBA.Full-strength MS medium containing different concentrations of IAA (0.5 and 1.0 mg/l), NAA (1.0 and 2.0 mg/l), and IBA (2.0 and 3.0 mg/l) produced enlarged callus-like structure on and around the root tip. This structure had an influence on reducing the number and length of roots compared to treatments without callus-like growth.Out of 102 plantlets which were planted in pots containing red soil, compost, and sand (2:1:1) placed in a green house, 60% survived after a month. Most of the dead plants were due to small root number or hairs, fungal contamination, and rotting. None of the plants survived in pots which were not covered by polyethylene bags and those containing unsterilized soil, compost, and sand.Aconitum heterphyllum  seeds. According to our results, the highest percentage of decontaminated seeds was observed at 15% sodium hypochlorite (Clorox), but low germination percentage. Guanih et al. [Dryobalanops lanceolata with 30% Clorox concentration for 5 min and cultured on MS medium showed less than 20% contamination, but sterilization of coated seeds with 50% Clorox concentration for more than 20 min broke the seed coat and reduced seed viability.The result showed that increasing Clorox concentration increases decontamination, but decreases viability of the seeds. The percentage of commercial Clorox needs to be varied depending on the species and environment . On the Andrographis pancuatanee, absence of significant correlation between contamination and germination percentages. Ghareeb and Taha [Antigonon leptopus explants using 10% Clorox for 3 min. In general, the lower the Clorox concentration, the higher the contamination and germination percentage.Among those Clorox concentrations, 10% Clorox resulted in 85% decontaminated seeds and the highest germination percentage (80%). Talei et al.  reportedand Taha  obtainedS. longipedunculata. In addition, Negash [Prunus africana, in a laboratory.Our result showed that the de-coated seeds showed better germination than coated seeds. As Zulu et al.  reported, Negash  reportedPsoralea corylifolia, was recorded on full-strength MS medium. Maślanka and Bach [S. longipedunculata using GA3.In the present study, the germination on full-strength MS medium was best among other half-strength germination media. According to Pandey et al. , the higand Bach  obtainedand Bach  suggesteand Bach  obtainedArnebia benthamii by removing the seed coat within mean germination time of around 4 days.Among all media, the highest germination percentage (100%) of seeds was observed within 7–10 days on MS medium, when the seeds were de-coated and transversally cut at the tip. This is partly due to the removing of the seed coat facilitated the uptake of water and cutting at the tip reduces the size to use the limited amount of moisture on the MS medium. Similarly, Ganaie et al.  reportedS. longipedunculata shoot explants. These treatments, with various combinations of BAP and TDZ resulted in shoot initiation. The percentage response of explants for shoot induction, shoot number, and shoot length varies according to the type and concentration of cytokinins used [In this study, the effect of plant growth regulators on shoot induction from shoot explants was investigated. Sixteen different treatments including the control were used to induce shoots from ins used . The resTrichosanthes dioica from nodal explants. The microshoots that resulted from MS medium containing 1.0 mg/l BAP were higher in number, but shorter in length than the shoots obtained from 1.0 mg/l BAP in combination with 0.5 mg/l TDZ. Ahmed and Anis [The highest percentage (87%) of explants survived on MS medium containing 1.0 mg/l BAP. Tiwari et al.  reportedand Anis  reportedProsopis cineraria. Similarly, Malik and Saxena [Phaseolus vulgaris.In general, the shoots were initiated better on MS medium containing BAP alone than MS medium containing TDZ alone or in combination with BAP. According to Kumar and Singh , BAP wasd Saxena  pointed Ceropegia mohanramii. In contrast, Fraternale et al. [Bupleurum fruticosum. According to Askari-Khorasgani et al. [Application of BAP in combination with IBA with different concentrations induced the highest shoot number and length per explant. From our result, the MS medium containing 1.5 mg/l BAP in combination with 0.1 mg/l IBA produced the highest mean number of shoots (8.50 ± 0.69). Medium supplemented with auxin at low concentrations in combination with cytokinin promote the growth and formation of new shoots, consequently, increasing multiplication rate . Adsul ee et al.  reportedi et al. , in compi et al.  stated ti et al. , 35. Brai et al.  reportedThe highest mean shoot length (2.11 ± 0.45 cm) was obtained on 2.5 mg/l BAP in combination with 0.1 mg/l IBA whereas the lowest shoot length (0.96 ± 0.40 cm) was recorded on MS medium containing 0.5 mg/l BAP in combination with 0.5 mg/l IBA. This is surprising as the concentration of BAP increased, shorter shoots are expected because of apical dominance indicating the present finding requires further investigation.Astragalus polemoniacus.On medium containing TDZ alone or in combinations with IBA, the highest mean number of shoots per explant (6.20 ± 0.43) was produced on MS medium containing 0.5 mg/l TDZ alone. Mirici  reportedPolygonatum verticillatum, suggested that the promoter effect of TDZ regarding growth is due to its own biological activities similar to N-substituted cytokinin or it may induce the synthesis or accumulation of an endogenous cytokinin. In case of TDZ and NAA combinations, the highest mean number of shoots (4.70 ± 0.32) was produced on MS medium containing 1.5 mg/l TDZ in combination with 0.5 mg/l NAAIn terms of length, the highest mean shoot length (1.55 ± 0.09 cm) was obtained on MS medium containing 0.5 mg/l TDZ. Aasim et al.  showed tGenerally, among all growth regulators used in shoot multiplication, the highest mean shoot number (8.5 ± 0.69) was obtained on MS medium containing 1.5 mg/l BAP in combination with 0.1 mg/l IBA. On the other hand, the lowest mean shoot number per explant (2.10 ± 0.12) was obtained on basal MS medium. MS medium containing 2.5 mg/l BAP in combination with 0.1 mg/l IBA produced the highest mean shoot length (2.11 ± 0.45 cm) while the shortest mean shoot length (0.16 ± 0.01 cm) was produced on MS medium containing 2.5 mg/l TDZ in combination with 0.01 mg/l NAA and this short shoot production could be due to higher inhibition of apical dominance by TDZ than BAP.Malus pumila [Andrographis macrobotrys using IAA. Contrary to the results of our study, Shen et al. [Casuarina cunninghamiana. It is important to work more on in vitro production of root and also root culture as the roots are used for medicinal purposes.Interestingly, full-strength MS medium containing 2.0 mg/l IAA, resulted in better rooting response (3.73 ± 0.69) as compared to IBA and NAA contrary to the most common half-strength MS medium used for rooting. IAA was found to be effective on rooting of s pumila . In addis pumila  reportedn et al.  pointed Ruta graveolens, the dead plantlets were due to improper development of root system in the culture. None of the plantlets survived in pots which were not covered with polyethylene bags.In this study, 60% plants survived in glasshouse after a month. This low survival percentage might be due to small root number or hairs, fungal contamination, and rotting. According to Bohidar et al.  who workSecuridaca longipedunculata is being destroyed because its root is used for medicinal purpose. Its natural regeneration is very difficult as the seed germination rate of the plant is very low. There is a need to produce superior genotypes of this plant to use its root for medicinal purposes and saving the natural stand. Therefore, the present study is very important for mass propagation of superior genotypes and conservation of this very important medicinal plant.


To analyze the detection rates of colorectal cancer (CRC) and polyps by population‐based screening in Guangzhou.From January 2011 to December 2015, the residents aged 30‐79 were selected for CRC screening. The residents were conducted Questionnaires and/or FOBT to assess high‐risk groups, the free colonoscopy examination was recommended, and the results were evaluated in detail.P = 0.019). And the detection rates increasing with age, for people over 60 years old, were obviously higher than those younger .There were 98 927 residents involving screening, 5306 high‐risk residents identified , and 4713 subjects underwent colonoscopy . CRC was seen in 55 individuals , and the detection rates in male were higher than in female (P = 2.64 × 10−14). For the detection rates of adenomas or advanced adenomas by age, the people over 40 years were higher than younger .The polyps were seen in 1458 (30.94%) cases， and 1420 subjects received pathological examination (adenomas 971 and non‐adenomatous polyps 449). Advanced adenomas were seen in 462 cases  and 509 cases of non‐advanced adenomas . For advanced adenomas, the detection rates in male were higher than female (14.20% vs 7.34%, For Guangzhou residents, the detection rates of CRC and adenoma were 1.17% and 20.60%. The detection rates of CRC increasing with age, for people over 60 years old, were obviously higher than those younger. But for people over 40 years, the detection rate of adenoma and advanced adenoma was higher than younger. So for people over 40 years, the CRC screening is recommended. A 2‐stage colorectal cancer screening program was conducted. For the first stage, the community residents were conducted Questionnaires and/or FOBT to assess high‐risk groups. The enrolled participants complete a Questionnaires, including the following: (a) For the following symptoms, there were at least two positive: chronic constipation, chronic diarrhea, mucus bloody stool, chronic appendicitis or appendectomy, chronic biliary tract disease, or cholecystectomy; (b) for every resident, FOBT were conducted twice by two different stool samples, and at least one positive; (c) the history of colonic polyps or CRC; (d) first‐degree relatives with colorectal cancer; and (e) first‐degree relatives of familial adenomatous polyposis. Patients with severe other diseases, such as severe heart disease, severe brain disease, severe lung disease, severe liver disease, severe kidney disease, and severe mental illness, were excluded. For every participating resident, the FOBT positive or at least one of the five Questionnaires positive was defined as high‐risk people.The above information was reviewed and collected by trained medical personnel. All data were inputted into a database with unique Resident Identity Card numbers. For every participating resident, the unique Resident Identity Card numbers were inputted into a database. For those high‐risk people, the free colonoscopy examination was recommended. The results of colorectal screening were evaluated in detail. The retrospective analysis protocol was approved by the Ethics Committee of Guangzhou First People's Hospital.2.2The FOBT reagents are purchased from WHPM Bioresearch and Technology Co., Ltd, which is One‐step Immunological Hemoglobin Test, the cutoff point is 0.2 µg hemoglobin/mL.2.3For all high‐risk people, the free colonoscopy examinations were recommended. Orally administered mannitol was used for colonic preparation before operations. For the defects, the size, number, polyp morphology, and the anatomical position were recorded. All colonoscopy examinations were conducted by the specialist endoscopists. The colonoscopies were performed in our two hospitals . All data were submitted to Yuexiu District Center for Disease Control and Prevention.2.4According to different guidelines, we selected the following key quality indicators in our colonoscopy screening: (a) informed consent; (b) rate of adequate bowel preparation ≥90%; (c) cecal intubation rate ≥90%; (d) adenoma detection rate ≥25%; (e) appropriate polypectomy technique ≥80%; (f) the average withdrawal time ≥8 minutes; and (g) serious adverse event, such as perforation or bleeding, must be recorded.2.5For lesions, the biopsy was taken and the pathology examinations were carried out.2.6P < 0.05.For multiple neoplasms, the most advanced one was recorded. Data were analyzed using the Statistical Package for Social Sciences (SPSS) software . The chi‐squared were used for categorical data. The statistical significance threshold was defined as 3From January 2011 to December 2015, a total of 98 927 participants entered population‐based colorectal cancer screening, 5306 (5.36%) were identified as high risk , and 4713  subjects underwent colonoscopy examinations, including 1690 males and 3023 females, from 30 to 79 (56.8 ± 8.5) years old. The cecum intubation rate was 97.54% (4597 of 4713), in this study, there was no perforation or no bleeding occurred.Totally, 55 cases 1.17%) had CRC and 1458 (30.94%) subjects had colorectal polyps. . And the detection rates increasing with age, for people aged 40 to 49, 50 to 59, 60 to 69, and 70 to 79 years old, the detection rate was 0.61%, 0.76%, 1.69%, and 3.03%, respectively ; hepatic flexure, 2 (3.63%); transverse colon, 5 (9.09%); splenic flexure, 2 (3.63%); descending colon, 5 (9.09%); sigmoid colon, 18 (32.73%); rectal, 16 (29.10%), respectively. Taken together, there were 25.45% (14 cases) located in the right‐sided colon, 45.45% (25 cases) located in the left‐sided colon, and 29.09% (16 cases) located in rectal .For 1458 subjects with colorectal polyps, 38 cases had no pathological examinations, 1420 received pathological examination, and 971 cases were adenomas , including 462 cases of advanced adenomas  and 509 cases of non‐advanced adenomas , 5.19% (24 cases), 12.77% (59 cases), 1.30% (6 cases), 13.64% (63 cases), 33.77% (135 cases), and 15.37% (71 cases), respectively , 422 were small adenomas (5‐9 mm), and 280 were large adenomas (≥10 mm).4CRC is the third most commonly diagnosed cancer in men and the second in women worldwide and the fourth cause of cancer death worldwide. It accounts for over 9% of all cancer incidences, with an estimated 1.4 million cases occurring in 2012.The global burden of colorectal cancer (CRC) is expected to increase by 60% to more than 2.2 million new cases and 1.1 million deaths by 2030.The distribution of CRC varies widely across the world, with almost 55% of the cases occurring in more developed countries, especially in high‐income countries,For twodecades, the CRC incidence rates have declined about 2% to 3% per year over the last 15 years in the United States, which is primarily associated with the increase in screening uptake and removal of precancerous adenomas.5 in 2000 to 34.0/105 in 2011, higher than the average in China for the same time.5 in 2000 to 23.2/105 in 2011, and for male, the crude incidence increased from 23.4/105 in 2000 to 37.4/105 in 2011. Our screening data support this result.In China, the CRC incidence and mortality rates are increasing rapidly for two decades. There is obvious diversity of age, gender, and geographical difference in CRC incidence and mortality rates. The well‐developed provinces in China presented high incidence and mortality rates. Furthermore, CRC incidence and mortality rates were higher in urban areas than in rural regions. In 2010, there were about 274 841 new cases diagnosed with CRC , with the crude incidence rate of 20.1/100 000, ranked the 6th in all cancer sites,The CRC incidence rates increase with age.Older age remains closely correlated with the incidence of adenomas,For the adenoma distribution within the colon, Lieberman et al found that in persons younger than 60 years, more than half of CRC were located in the distal colon, whereas in persons aged 60 years and older, a slightly higher percentage of CRC were found in the proximal colon.In conclusion, our findings indicate that in Yuexiu District, Guangzhou, the detection rates of CRC, adenoma, and advanced adenoma are higher in male than in female. CRC screening is recommendation for the residents, especially for those over 40 years in male.None declared.


M1 and M3 were identified as important metabolites in vitro for either antihypertension activity or the major metabolite production. In order to obtain a pharmacokinetic profile of both TJ0711 and its metabolites, a rapid, selective, and reliable LC-MS/MS method was developed and validated for simultaneous determination of TJ0711 and two metabolites in beagle dog plasma via efficiently separating two interferential metabolites M16 and M4 from M1 and M3, respectively. Chromatographic separation was achieved on a Waters CORTECS C18+ column . The mass spectrometric detection was carried out in positive ion MRM mode with ESI+ source. Protein precipitation was used in sample preparation and provided good recovery without a matrix effect. Good linearity was observed at the ranges of 0.5–100 ng/mL for TJ0711 and M3, 0.1–20 ng/mL for M1. Additional validation results were within the acceptance limits followed U.S. FDA guidelines for bioanalytical method validation. This method was successfully applied to an intravenous infusion pharmacokinetic study of TJ0711 at dosing rates of 3, 6, and 12 µg/kg/min in anesthetized beagle dogs for the first time. TJ0711 and its two metabolites exhibited effective proportionality in the dosage of 3 to 12 µg/kg/min. Neither TJ0711 nor its metabolites showed significant differences in pharmacokinetic parameters such as t1/2, CL, and ssV among three dose groups.TJ0711∙HCl, which is a novel α In ttivities  and 45 mtivities . Due to In in vitro metabolism studies, a total of 33 metabolites were found including 19 phase I and 14 phase II metabolites . MoreoveSeveral precious analytical methods have been reported for TJ0711 by our group using high-performance liquid chromatography (HPLC)-UV  and HPLC2) tubes  and centrifuged at 1200× g for 10 min at 4 °C to obtain blank plasma. The blank plasma was then stored at −80 °C until used.TJ0711 hydrochloride, M1 hydrochloride, and M3 were synthesized in our laboratory. The purity of TJ0711, M1, and M3 was >99.8%, >97.3%, and >99.5%, respectively, which was measured by a high-performance liquid chromatography (HPLC) system with ultraviolet (UV) detector. Propranolol hydrochloride  was obtained from the National Institutes for Food and Drug Control . The chemical structures of TJ0711, two metabolite M1, M3, and IS are shown in ® triple quadrupole mass spectrometer  equipped with an electrospray ionization (ESI) source was used throughout the study. Chromatographic separation was achieved on a Waters CORTECS C18+ column  equipped with a guard column . The column temperature was maintained at 40 °C. The mobile phase consisted of 0.1% (v/v) formic acid in water (Solvent A) and ACN (Solvent B). The flow rate was maintained at 0.5 mL/min. The following gradient conditions were used: 0–2 min, 15–20% B, 2–4 min, 20% B, 4–5 min, 80% B, 5–6 min, 15% B. The injection volume was 10 μL and the auto sampler temperature was set at 4 °C. A Shimadzu Prominence UFLC system  coupled with an API 4000 QTrapMass spectrometric detection was performed in a positive electrospray ionization (ESI) mode. Quantification was conducted using a multiple reaction monitoring (MRM) mode and the following MRM transitions 376.2→252.2, 362.0→238.0, and 260.1→116.1 were used for TJ0711 (M3 yielded the same precursor and product ions as TJ0711), M1 and IS, respectively, with a dwell time of 150 ms for each MRM transition. The mass spectrometric parameters including de-clustering potential (DP), collision energy (CE), entrance potential (EP), and cell exit potential (CXP) for each compound were optimized as follows: DP: 91 V, CE: 29 V, EP: 10 V, CXP: 6 V for both TJ0711 and M3; DP: 91 V, CE: 37 V, EP: 10 V, CXP: 8 V for M1; DP: 62 V, CE: 25 V, EP: 10 V, CXP: 8 V for IS. The source parameters were optimized as follows: ion spray voltage, 5500 V, source temperature, 600 °C, curtain gas, 25 psi, nebulizer gas, 40 psi, auxiliary gas, 50 psi, collision activated dissociation gas, high, interface heater, on. Analyst 1.6.1 software  was used for the instrument control, data acquisition, and processing.TJ0711, M1, M3, and IS stock solutions were prepared using 50% ACN (v/v). The concentrations of TJ0711, M1, M3, and IS were corrected by the purity and salt correction factor of each compound. The corrected concentration was 10 mg/mL for TJ0711 and 5 mg/mL for the others. Calibration curve and quality control working solutions were prepared by serially diluting stock solutions separately in 50% ACN, all of which contained appropriate concentrations of each analyte. Furthermore, 20 μL working solution was spiked with 480 μL blank dog plasma to obtain the required final concentrations of the calibration standard (CS) and quality control (QC) samples. The final concentrations of all analytes of interest in CS and QC samples are listed in g for 10 min at 4 °C, 480 μL of the supernatant was aspirated and evaporated using an SPD1010 SpeedVac Concentrator  at 45 °C, 5.1 Vac for 2.5 h. The residue was reconstituted in 80 μL of 15% ACN in water.All plasma samples including CS samples, QC samples, and plasma samples from pharmacokinetic study were prepared by protein precipitation. In addition, 100 μL plasma was spiked into 500 μL ACN, which contained 20 ng/mL IS. After vortex for 5 min and centrifugation at 16,000× 2 plasma samples  were processed in order to investigate the specificity and selectivity of this method. The peak area of interference should be ˂20% of the area of analytes in LLOQ samples and ≤5% for IS. LOD was defined as the concentration of which the signal to noise ratio (S/N) was larger than 3. LLOQ was defined as the lowest concentration of the calibration curve that yielded a peak with an S/N ≥ 10 while the accuracy should be 80% to 120% and precision should not exceed 20%. The linearity of the calibration curves was evaluated using eight calibration standards for each analyte in three different batches on three separate days. The calibration curves were plotted using the ratio of each analyte peak area over the internal standard peak area versus the nominal concentration using a weighting factor of 1/x2. To assess intra-assay and inter-assay accuracy and precision, LLOQ and three concentration QC level  samples were prepared along with the calibration curves on three separate days. According to the guidelines of the FDA, the error of accuracy and coefficient of variation (CV) should not exceed 15% for all CS and QC samples, except for the LLOQ (<20%). The matrix effects were evaluated by comparing the peak area of analytes of QC samples prepared from six-individual dog blank plasma with a peak area of neat solutions. Relative recovery was assessed by calculating the analyte peak area in spiked samples before extraction over the analyte peak area in spiked samples after extraction. Carry-over was investigated by running a double blank sample after the upper limit of quantitation (ULOQ) sample. The dilution integrity was evaluated by diluting dog plasma at the concentration 5-fold or 10-fold HQC with blank plasma using a ratio of 1:4 or 1:9, respectively. In this study, the stability of analytes was evaluated in both neat solutions and biological specimens. The stock solutions and working solutions were determined after 24 h storage in room temperature or 34 days storage in 4 °C. Besides, the stability of analytes in dog plasma was investigated by determining the concentrations of LQC and HQC samples after they had been kept at room temperature for 24 h, at −80 °C for 30 days, or after three freeze/thaw cycles. The IS was spiked after the storage for stability determination. The stability of TJ0711 and its metabolites in the processed samples was evaluated at auto sampler temperature of 4 °C for 24 h. For this situation, the IS has been added with a precipitation reagent for sample clean-up. The analytes were considered to be stable in biological specimens if the concentrations determined were 85% to 115% of the original concentration. The stability of analytes in whole blood was assessed as well.The method validation was followed by the United States Food and Drug Administration guidance for bioanalytical method validation . Selecti2 tubes and centrifuged at 1200× g for 10 min at 4 °C. The plasma was isolated into EP tubes and stored at −80 °C until analyzed.The validated method was applied to the pharmacokinetic study of TJ0711 in beagle dogs. The Animal Ethics Committee in Tongji Medical College, Huazhong University of Science and Technology  approved this study (Ref No. 00221434). Six beagle dogs -2016-0057) weighing 10 ± 2 kg were anesthetized using isoflurane  with Animal Anesthesia Ventilator . Thereafter, TJ0711 hydrochloride, prepared in normal saline, was administrated by intravenous infusion at a dose rate of 3 μg/kg/min. After every 10 day washout period, another dose (6 or 12 μg/kg/min) was administrated. Approximately 1 mL blood samples were withdrawn via the cephalic vein at 0, 10, 20, 40, 60, 90, 120, 180, 240, 260, 280, 300, 360, 420, 480, and 600 min for 3 and 6 µg/kg/min and at 0, 10, 20, 40, 60, 90, 120, 180, 240, 260, 280, 300, 360, 480, 600, and 720 min for 12 μg/kg/min after dosing. Blood samples were collected in EDTA-KmaxC) and time to reach maxC (maxT) were obtained directly from plasma concentration data. The area under the concentration versus time curve (including AUCt0– and AUC0–∞), half-life (t1/2), steady-state volume of distribution (ssV), and clearance (CL) were calculated by WinNonlin 6.4 software. The lambda z ranges were calculated by WinNonlin software automatically and 3 to 6 points were used for half life calculation in individual dogs. Linear trapezoidal method was used for calculation of AUC. Then a power function model was applied to the pharmacokinetic parameters (maxC and AUC0–∞) over the dose range by MATLAB R2017a software  to assess the dose proportionality. The power function model was presented as PK = α × (Dose)β. It is assumed that the natural ln-transformed pharmacokinetic parameters are linearly related to natural ln-transformed dose: ln(PK) = α + β × ln(Dose) [The pharmacokinetic parameters were estimated using a non-compartmental model with Phoenix WinNonlin 6.4 software . Maximum plasma concentration (ln(Dose) . The slo+ column. Therefore, 0.1% formic acid was chosen as an additive in aqueous phase due to higher sensitivity and lower background interference. It was worth noting that, except M3, there was another metabolite (M4), which yielded the same precursor and product ions as the parent, while M16 yielded the same precursor and product ions as M1 [In our study, chromatographic conditions, mass spectrometric (MS) procedures, and a sample pretreatment method were optimized to obtain symmetrical peak shape, maximize the MS response, and achieve more accurate quantification. The chromatographic conditions including organic phase, additives, and gradient profiles were investigated. ACN was chosen because of the better chromatographic separation and improved peak shape compared to methanol. In previous LC-MS/MS method, isocratic elution was applied using ammonium formate solution as aqueous phase . Unfortuns as M1 . GradienFor sample clean-up, two different types of sample pretreatment methods, liquid-liquid extraction (LLE), and protein precipitation (PPT) were evaluated. In our study, five extraction solvents including ethyl acetate, hexane, chloroform, methyltertiarybutyl ether (MTBE), and diethyl ether were tested. Due to alkalescence of the analytes and IS, 20 µL of 0.5 M sodium hydroxide was used to enhance the extraction recovery for both analytes and IS. However, M3 has a carboxylate radical and, thus, existed as an ionic type under an alkaline condition. Therefore, the extraction process was optimized by adding sodium hydroxide and/or hydrochloric acid . RelativDouble blank samples from six individual dogs were compared with the LLOQ sample. The typical chromatograms of double blank sample, the LLOQ sample, and a sample collected 120 min after dosing are shown in The LOD of TJ0711, M1, and M3 were 0.01 ng/mL of which S/N was larger than 3. The LLOQ was 0.5, 0.1, and 0.5 ng/mL for TJ0711, M1, and M3, respectively. The accuracy and precision of LLOQ met acceptance criteria and the signal-to-noise values of each analyte in LLOQ were greater than 10, which indicated that the established method was sensitive for the quantification and pharmacokinetic analysis of TJ0711, M1, and M3 in dog plasma.2 to assess linearity. The range of linearity for TJ0711 and M3 was 0.5 to 100 ng/mL and for M1 was 0.1 to 20 ng/mL. Eight calibration standards were performed in triplicate on three separated days. In line with the requirement, eight sets of the calibrators were within <15% (<20% for the LLOQ) of the nominal concentrations. The linear range, regression equations, and correlation coefficients are listed in calculated values of 23.0, 327.3, and 0.98 for TJ0711, M1, and M3, respectively, and tabulated F0.05  value was found to be 6.61, which indicates that TJ0711, and M3 calibration standards were fitted better by a quadratic function. For example, typical calibration curves for TJ0711 and M3 were y = −8.25 × 10−5x2 + 0.056x + 5.87 × 10−4 and y = −5.47 × 10−5x2 + 0.0443x + 5.45 × 10−5, respectively. These equations had a significantly smaller coefficient (−5 × 10−5~−9 × 10−5) for the variable term of “x2” compared to the coefficient (×10−2) for the variable term of “x”. However, in bioanalytical studies, a linear calibration function was commonly used for calibration curves. Accuracy and precision results calculated by linear calibration fitting were not compromised and also met FDA bioanalysis guidance acceptance criteria. Therefore, a linear calibration function rather than a quadratic function was finally chosen. In addition, both of the function y = ax + b and y = ax were used for the calibration curve assessment of M1. The intercepts of individual calibration curve were calculated by Analyst Software 1.6.1. There were no significant differences (p > 0.05) between the intercepts of two equations. Thus, the function y = ax instead of y = ax + b was employed for the corrected calibration equation of M1 .A double blank sample was run after the ULOQ sample to observe carry-over. No peak was found in the double blank sample after the ULOQ sample for both analytes and IS, which verified that there was no carry-over in the method.The stability of TJ0711, M1, and M3 in dog plasma was evaluated using LQC and HQC samples and the results are presented in AUCt0– of M1 did not exceed 5% of TJ0711, which might relate to the weak metabolic stability of M1 in dog liver microsomes. In the in vitro metabolic stability of M1, the remaining percentage of M1 was above 70% after 60 min incubation in 0.25 mg/mL human liver microsomes, while the elimination of M1 was faster in dog liver microsomes, which indicates significant differences between species in metabolic stability of M1 [maxC of TJ0711 and AUC0–∞ of TJ0711). The proportionality constant β (95% CI) for maxC and AUC0–∞ of TJ0711 were 0.9819 (0.7745–1.1890) and 1.0050 (0.7958–1.2140), respectively, which indicates linear does-proportionality. Moreover, M1 and M3 also exhibited good does-proportionality in the dosage of 3–12 µg/kg/min  compared to our previous study [According to the previous pharmacokinetic/pharmacodynamic studies after a single intravenous injection of TJ0711 in conscious beagle dogs (data not published), the blood pressure decreased by 20% when plasma TJ0711 concentration reached 100 ng/mL, which was recognized as an effective antihypertensive concentration. Based on clearance and half life results of TJ0711, 6 µg/kg/min was selected as a medium dosing rate of our study. Then, 3 µg/kg/min and 12 µg/kg/min were chosen to be low and high dosing rate. The mean measured plasma concentration versus time profiles of TJ0711, M1, and M3 are presented in ty of M1 . A powerus study . In the us study . The difIn our study, a rapid, selective, and reliable LC-MS/MS method was developed and validated for the simultaneous quantitation of TJ0711 and two metabolites (M1 and M3) in beagle dog plasma. Due to the low extraction recovery of M3 in five extraction agents under various extraction conditions of LLE, PPT followed by evaporation and reconstitution was used to prepare plasma samples, which leads to a high MS response and no matrix effect. The validated method was successfully applied to pharmacokinetic study in beagle dogs following intravenous infusion of TJ0711∙HCl. The PK profiles of TJ0711 and its two metabolites followed linear process between the dosing rates of 3 to 12 µg/kg/min. Furthermore, the differences of the PK process were found between conscious and anesthetized dogs, which indicates that caution should be taken in clinical use of TJ0711 under conscious and anesthetic states.