Abstract Context Dacomitinib and poziotinib, irreversible ErbB family blockers, are often used for treatment of non-small cell lung cancer (NSCLC) in the clinic. Objective This study investigates the effect of dacomitinib on the pharmacokinetics of poziotinib in rats. Materials and methods Twelve Sprague–Dawley rats were randomly divided into two groups: the test group (20 mg/kg dacomitinib for 14 consecutive days) and the control group (equal amounts of vehicle). Each group was given an oral dose of 10 mg/kg poziotinib 30 min after administration of dacomitinib or vehicle at the end of the 14 day administration. The concentration of poziotinib in plasma was quantified by UPLC-MS/MS. Both in vitro effects of dacomitinib on poziotinib and the mechanism of the observed inhibition were studied in rat liver microsomes and human liver microsomes. Results When orally administered, dacomitinib increased the AUC, T max and decreased CL of poziotinib (p < 0.05). The IC50 values of M1 in RLM, HLM and CYP3A4 were 11.36, 30.49 and 19.57 µM, respectively. The IC50 values of M2 in RLM, HLM and CYP2D6 were 43.69, 0.34 and 0.11 µM, respectively, and dacomitinib inhibited poziotinib by a mixed way in CYP3A4 and CYP2D6. The results of the in vivo experiments were consistent with those of the in vitro experiments. Conclusions This research demonstrates that a drug–drug interaction between poziotinib and dacomitinib possibly exists when readministered with poziotinib; thus, clinicians should pay attention to the resulting changes in pharmacokinetic parameters and accordingly, adjust the dose of poziotinib in clinical settings.

Fuzi decoction was commonly used for the treatment of Yang exhaustion and inward invasion of cold-damp according to the theory of Traditional Chinese Medicine (TCM). In the study, UPLC-MS/MS was used to establish a selective and sensitive method to determine three main components in Fuzi decoction- benzoylaconine, benzoylhypaconine and atractylenolide I with carbamazepine as IS (internal standard) in plasma of rat. The sample was prepared by precipitating acetonitrile as a precipitant to precipitate the protein. Separation of benzoylaconine, benzoylhypaconine, atractylenolide I and carbamazepine were performed on a CORTECS UPLC C18 column (2.1 x 50 mm, 1.6 mu m). The mobile phase (acetonitrile: water of 0.1% formic acid) by gradient elution was set a flow rate of 0.4 mL/min. The ions of target fragment, 604.3 -> 105 m/z for benzoylaconine, 574.3 -> 105 m/z for benzoylhypaconine, 231.04 -> 185.11 m/z for atractylenolide I and 237 -> 194.1 m/z for IS, could quantify the electrospray ionization which positived of multiple reaction monitoring (MRM) mode. The linear calibration curve of the concentration range were 0.5-100 ng/mL for benzoylaconine and atractylenolide I, with a LLOQ (lower limit of quantification) of 0.1 ng/mL and 1-250 ng/mL for benzoylhypaconine, with a LLOQ of 0.2 ng/mL. RSD of inter-day and intra-day precision were both no more than 7.42% with the accuracy ranged from 98.81% and 106.05%. The average recovery of BAC, BHC and ALT were 98.87%, 102.24% and 101.33%. The developed and validated method was perfectly used in the pharmacokinetic study of Fuzi decoction after oral administration in rats.

PURPOSE:Avitinib is the first third-generation epithelial growth factor receptor (EGFR) inhibitor independently developed in China and is mainly used for treating non-small cell lung cancer. However, pharmacokinetic details are limited. This study explored the in vivo and in vitro effects of avitinib on cytochrome CYP450 enzymes metabolic activity.METHODS:A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determining six probe substrates and their metabolites. Avitinib influence on activity levels of CYP isozymes was examined in vitro using human and rat liver microsomes (HLMs/RLMs). For in vivo studies, rats were pretreated with 30 mg/kg avitinib once daily for 7 days (avitinib multiple-doses group), 30 mg/kg avitinib on day 7 (avitinib single-dose group), or an equivalent amount of CMC-Na once daily for 7 days (control group), followed by intragastrical administration of the probe substrates (1 mg/kg tolbutamide and 10 mg/kg phenacetin, bupropion, chlorzoxazone, dextromethorphan, and midazolam). Plasma pharmacokinetics and IC50 values of the probe substrates were then compared. Pharmacokinetic parameters were determined using non-compartmental analysis implemented in a pharmacokinetic program.RESULTS:In vitro experiments revealed different inhibitory effects of avitinib on the six probe substrates with various IC50 values (bupropion, 6.39/22.64 μM; phenacetin, 15.79/48.36 μM; chlorzoxazone, 23.15/57.09 μM; midazolam, 27.64/59.6 μM; tolbutamide, 42.18/6.91 μM; dextromethorphan, 44.39/56.57 μM, in RLMs and HLMs respectively). In vivo analysis revealed significant differences (P <0.05) in distinct pharmacokinetic parameters (AUC(0-t), AUC (0-∞), Cmax, MRT(0-t), MRT (0-∞), and CLz/F) for the six probe substrates after avitinib pretreatment.CONCLUSION:A sensitive and reliable UPLC-MS/MS method was established to determine the concentration of six probe substrates in rat plasma. Avitinib had inhibitory effects on CYP450 enzymes, especially cyp2b1, cyp1a2 in RLMs, CYP2C9 in HLMs, and cyp1a2, cyp2b1, cyp2d1, and cyp2e1 in vivo. Our data recommend caution when avitinib was taken simultaneously with drugs metabolized by CYP450 enzymes.

1Comprehensive Breast Health Center, Department of Thyroid and Breast Surgery, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, 323000, People’s Republic of China; 2Department of Pharmacy, Ningbo First Hospital, Ningbo, 315010, Zhejiang, People’s Republic of China; 3Department of Pharmacy, Ningbo Municipal Hospital of Traditional Chinese Medicine, Ningbo, 315010, Zhejiang, People’s Republic of China; 4Department of Radiology, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, 323000, People’s Republic of China

Poziotinib is an orally active, irreversible, pan-HER tyrosine kinase inhibitor used to treat non-small cell lung cancer, breast cancer, and gastric cancer. Poziotinib is currently under clinical investigation, and understanding its drug-drug interactions is extremely important for its future development and clinical application. The cocktail method is most suitable for evaluating the activity of cytochrome P450 enzymes (CYPs). As poziotinib is partially metabolized by CYPs, cocktail probes are used to study the interaction between drugs metabolized by each CYP subtype. Midazolam, bupropion, dextromethorphan, tolbutamide, chlorzoxazone, phenacetin, and their metabolites were used to examine the effects of poziotinib on the activity of cyp1a2, 2b1, 2d1, 2c11, 2e1, and 3a1/2, respectively. The in vitro experiment was carried out by using rat liver microsomes (RLMs), whereas the in vivo experiment involved the comparison of the pharmacokinetic parameters of the probes after co-administration with poziotinib to rats to those of control rats treated with only probes. UPLC-MS/MS was used to detect the probes and their metabolites in rat plasma and rat liver microsomes. The in vitro results revealed that the half-maximal inhibitory concentration values of bupropion and tolbutamide in RLMs were 8.79 and 20.17 μM, respectively, indicating that poziotinib showed varying degrees of inhibition toward cyp2b1 and cyp2c11. Poziotinib was a competitive inhibitor of cyp2b1 and cyp2c11, with Ki values of 16.18 and 17.66 μM, respectively. No time- or concentration-dependence of inhibition by poziotinib was observed toward cyp2b1 and cyp2c11 in RLMs. Additionally, no obvious inhibitory effects were observed on the activity of cyp1a2, cyp2d1, cyp2e1, and cyp3a1/2. In vivo analysis revealed that bupropion, tolbutamide, phenacetin, and chlorzoxazone showed significantly different pharmacokinetic parameters after administration ( p < 0.05); there was no significant difference in the pharmacokinetic parameters of dextromethorphan and midazolam. These results show that poziotinib inhibited cyp2b1 and cyp2c11, but induced cyp1a2 and cyp2e1 in rats. Thus, poziotinib inhibited cyp2b1 and cyp2c11 activity in rats, suggesting the possibility of interactions between poziotinib and these CYP substrates and the need for caution when combining them in clinical settings.

1Department of Orthopaedics, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, Zhejiang 323000, People’s Republic of China; 2The Laboratory of Clinical Pharmacy, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, Zhejiang 323000, People’s Republic of China; 3School of Pharmaceutical Science, Guangdong Provincial Key Laboratory of New Drug Screening, Southern Medical University, Guangzhou 510515, People’s Republic of China

1The Laboratory of Clinical Pharmacy, The Sixth Affiliated Hospital of Wenzhou Medical University, The People’s Hospital of Lishui, Lishui, Zhejiang 323000, People’s Republic of China; 2The Key Laboratory of Geriatrics, Beijing Institute of Geriatrics, Beijing Hospital, National Center of Gerontology, National Health Commission, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, People’s Republic of China

Background Diazepam is a benzodiazepine drug used to treat anxiety, insomnia, and muscle spasms. Imperatorin is a phytochemical isolated from medicinal plants and is widely used in herbal medicine. The aim of this study was to investigate the interactions between imperatorin and diazepam in vitro and in vivo and to provide evidence-based guidance for the safe clinical use of the drug. Methods In vitro inhibition of imperatorin was assessed by incubating rat liver microsomes with diazepam to determine IC50 values and the type of inhibition. For in vivo assessment, six rats were pretreated with 50 mg/kg imperatorin for two weeks, six were administered saline, and a single dose of 10 mg/kg diazepam was administered orally to both groups 30 min after the administration of imperatorin. Results Imperatorin inhibited the in vitro metabolism of diazepam via the competitive mechanism of CYP450. The IC50 values of imperatorin to nordazepam and temazepam were 1.54 μM and 1.80 μM, respectively. The inhibitory constant values for temazepam and nordazepam were 1.24 μM and 1.29 μM, respectively. Long-term administration of imperatorin significantly increased the AUC(0-12h), AUC(0-∞), and Cmax of diazepam, while Vz/F and CLz/F were decreased significantly (P < 0.05). In turn, the AUC(0-12h), AUC(0-∞), and Cmax of nordazepam and temazepam decreased significantly, and Vz/F and CLz/F increased significantly (P < 0.05). Conclusions This study demonstrates that imperatorin inhibits the metabolism of diazepam both in vitro and in vivo. These results indicated that more attention should be paid when taking diazepam together with food or herbs containing IMP, although further investigation is still needed.

Botrychium ternatum was commonly used for the treatment of lung diseases according to the theory of Traditional Chinese Medicine (TCM). An ultra-liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of three main components in Botrychium ternatum decoction: luteolin, kaempferol, and quercetin in rat plasma, using cynaroside as an internal standard (IS). The samples were chromatographed on a CORTECS UPLC C18 column (2.1 x 50 mm, 1.6 mu m) by a mobile phase consisting of acetonitrile and water (0.1% formic acid) at a flow rate of 0.4 mL/min. The protonated ions of analytes were detected in negative ionization in multiple reaction monitoring mode (MRM). The ions of target fragment were 284.98 -> 0133.03 m/z for luteolin, 284.97 -> 092.94 m/z for kaempferol, 301.12 -> 0151.05 m/z for quercetina, and 447.05 -> 0284.96 m/z for IS. The linear calibration curve of the concentration range were 0.5 to 100 ng/ml. for luteolin, 0.2-50 ng/mL for kaempferol, and 1-250 ng/mL for quercetin, with a LLOQ (lower limit of quantification) of 0.2 ng/mL for quercetin and 0.2 ng/mL for luteolin and kaempferol. RSD of inter-day and intra-day precision were both no more than 9.77% with the accuracy ranged from 95.54 to 106.05%. The average recovery of luteolin, kaempferol. and quercetin ranged from 89.53 to 114.02%. The developed and validated method was perfectly used in the pharmacokinetic study of Botrychium ternatum decoction after oral administration in rats.

Sarecycline is a narrow-spectrum antibiotic for the treatment of acne, which is a chronic inflammatory disease of the hair follicle sebaceous glands. In the study, UPLC-MS/MS was used to establish a rapid and accurate analytical method. The sarecycline was determined with poziotinib as internal standard (IS) in rat plasma. An ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm) could performe chromatographic separation with the mobile phase (methanol: water of 0.1% formic acid) with gradient elution. The ions of target fragment were m/z 488.19→410.14 for sarecycline and m/z 492.06→354.55 for poziotinib, which could quantify the electrospray ionization of positive multiple reaction monitoring (MRM) mode. The linear calibration curve of the concentration range was 1–1,000 ng/mL for sarecycline with a lower limit of quantification (LLOQ) of 1 ng/mL. The mean recovery was between 82.46 and 95.85% for sarecycline and poziotinib in rat plasma. RSD for precision of inter-day and intra-day were between 3.24 and 13.36%, and the accuracy ranged from 105.26 to 109.75%. The developed and validated method was perfectly used in the pharmacokinetic study and bioavailability of sarecycline after intravenous and oral administration in rats.

A selective liquid chromatographic-mass spectrometric method (LC-MS/MS) has been established and validated for simultaneous determination of melatonin and its active metabolite in human plasma. Plasma samples were extracted by one step protein precipitation procedure and separated on a Acquity BEH C18 column (2.1 x 50 mm, 1.7 in particle size), with acetonitrile-0.1% formic acid solution as mobile phase at flow rate of 0.4 ml/min. The linear range was 1-500 ng/mL for melatonin and 0.1-50.0 ng/mL for its active metabolite, with lower limit of quantitation of 1 ng/mL and 0.1 ng/mL, respectively. Intra- and inter day precision and accuracy met the requirements, with the extraction recoveries 90.99-94.49% and 86.01-9432% for the two analytes, respectively. The validated method was successfully applied to a pharmacokinetic study of melatonin and its active metabolite in rat after oral administration and intravenous of melatonin.

Purpose The purpose of the present study was to investigate the effects of vonoprazan on the pharmacokinetics of venlafaxine in vitro and in vivo. Methods The mechanism underlying the inhibitory effect of vonoprazan on venlafaxine was investigated using rat liver microsomes. In vitro, the inhibition was evaluated by determining the production of O-desmethylvenlafaxine. Eighteen male Sprague–Dawley rats were randomly divided into three groups: control group, vonoprazan (5 mg/kg) group, and vonoprazan (20 mg/kg) group. A single dose of 20 mg/kg venlafaxine was administrated to rats orally without or with vonoprazan. Plasma was prepared from blood samples collected via the tail vein at different time points and concentrations of venlafaxine and its metabolite, O-desmethylvenlafaxine, were determined by ultra-performance liquid chromatography-tandem mass spectrometry. Results We observed that vonoprazan could significantly decrease the amount of O-desmethylvenlafaxine (IC50 = 5.544 μM). Vonoprazan inhibited the metabolism of venlafaxine by a mixed inhibition, combining competitive and non-competitive inhibitory mechanisms. Compared with that in the control group (without vonoprazan), the pharmacokinetic parameters of venlafaxine and its metabolite, O-desmethylvenlafaxine, were significantly increased in both 5 and 20 mg/kg vonoprazan groups, with an increase in MRO-desmethylvenlafaxine. Conclusion Vonoprazan significantly alters the pharmacokinetics of venlafaxine in vitro and in vivo. Further investigations should be conducted to check these effects in humans. Therapeutic drug monitoring of venlafaxine in individuals undergoing venlafaxine maintenance therapy is recommended when vonoprazan is used concomitantly.

Lusutrombopag is a second oral thrombopoietin (TPO) receptor agonist that selectively acts on human TPO receptors. In the study, UPLC-MS/MS was used to establish a selective and sensitive method to determine lusutrombopag with poziotinib as IS (internal standard) in rat plasma. Samples were prepared by precipitating protein with acetonitrile as a precipitant. Separation of lusutrombopag and poziotinib was performed on a CORTECS UPLC C18 column (2.1 ∗ 50 mm, 1.6 μm). The mobile phase (acetonitrile and water containing 0.1% formic acid) with gradient elution was set at a flow rate of 0.4 ml/min. The mass spectrometric measurement was conducted under positive ion mode using multiple reaction monitoring (MRM) of m/z 592.97 ⟶ 491.02 for lusutrombopag and m/z for poziotinib (IS) 492.06 ⟶ 354.55. The linear calibration curve of the concentration range was 2–2000 ng/ml for lusutrombopag, with a lower limit of quantification (LLOQ) of 2 ng/ml. RSD of interday and intraday precision were both no more than 9.66% with the accuracy ranging from 105.82% to 108.27%. The extraction recovery of lusutrombopag was between 82.15% and 90.34%. The developed and validated method was perfectly used in the pharmacokinetic study of lusutrombopag after oral administration in rats.

Diphenoxylate has a pharmacological effect similar to that of opioids, has the efficacy of an opioid active substance, and compound diphenoxylate tablets are easier to obtain as prescription drugs than other opioid preparations, so there are some abused by opioid addicts. In this study, we developed a serum metabolomic method by gas chromatography-mass spectrometry (GC-MS) to evaluate the effect of diphenoxylate treated on rats. The rats were divided into three groups, the control group, Low-dose (12 mg/kg) diphenoxylate treated group, Medium-dose (24 mg/kg) diphenoxylate treated group, High-dose (48 mg/kg) diphenoxylate treated group, diphenoxylate and saline intraperitoneal injection for 7 days. Serum samples were collected from the rats from three groups at 8:00 am after 7 day. The sample preparation for GC-MS analysis was according to our previous work. According to the serum metabolomics results, diphenoxylate treated rats could be distinguished from control group, and High-dose diphenoxylate treated group could be separated from Low-dose and Medium-dose diphenoxylate treated rats. Compared to the control group, Myo-Inositol decreased in Low-dose and High-dose diphenoxylate treated group; while L-norleucine, L-isoleucine, serine, 2,3,4-trihydroxybutyric acid, alanine, d-glucose, tyrosine, beta-D-glucopyranuronic acid, oleic acid, 9,12-octadecadienoic acid increased in three dose diphenoxylate treated group. The changes of metabolites increased or decreased, indicating that diphenoxylate treated rats induced amino acid metabolism, energy metabolism perturbations in rats. The results indicate that metabolomic method by GC-MS may be useful to elucidate diphenoxylate treated on rats.

Purpose: The purpose of this study was to examine the effects of voriconazole on the pharmacokinetics of vonoprazan. Methods: Fifteen Sprague-Dawley rats were randomly divided into three groups: five rats in each group, including control group, single-dose group (a single dose of 30 mg/kg of voriconazole), and multiple-dose group (multiple doses of 30 mg/(kg.day) per dose of voriconazole). Each group of rats was given an oral dose of 10 mg/kg vonoprazan 30 min after the administration of voriconazole or vehicle. After the oral administration of vonoprazan, 50 mu L of blood was collected into 1.5-mL heparinized tubes via the caudal vein. The concentration of vonoprazan in plasma was quantified by ultra-performance liquid chromatography/tandem mass spectrometry. Both in vitro effects of voriconazole on vonoprazan and the mechanism of the observed inhibition were studied in rat liver microsomes. Results: When orally administered, voriconazole increased the area under the plasma concentration-time curve (AUC), prolonged the elimination half-life (t(1/2)), and decreased the clearance (CL) of vonoprazan; there was no significant difference between the single-dose and multiple-dose groups. Voriconazole inhibited the metabolism of vonoprazan at an IC50 of 2.93 mu M and showed mixed inhibition. The results of the in vivo experiments were consistent with those of the in vitro experiments. Conclusion: Our findings provide the evidence of drug-drug interactions between voriconazole and vonoprazan that could occur with pre-administration of voriconazole. Thus, clinicians should pay attention to the resulting changes in pharmacokinetic parameters and accordingly, adjust the dose of vonoprazan in clinical settings.

BackgroundAvitinib is one type of the third‐generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for the treatment of non‐small cell lung cancer (NSCLC) with EGFR mutations. The purpose of this study was to investigate the effect of avitinib on the pharmacokinetics of osimertinib, one FDA approved third‐generation TIKI, both in vitro and in vivo.MethodsThe in vitro metabolic stability and inhibitory effect of avitinib on osimertinib were assessed with rat liver microsomes (RLM) to determine its IC50 values. For the in vivo study, 18 Sprague‐Dawley rats were randomly divided into three groups: the avitinib multiple dose group (30 mg/kg avitinib once daily for seven days), the avitinib single dose group (PEG200 once daily for six days and a dose of 30 mg/kg avitinib in PEG200 on day 7) and the control group (equal amounts of PEG200 once daily for seven days). Next, all rats were given osimertinib at a dosage of 10 mg/kg. UPLC/MS‐MS was used for the determination of the concentration of osimertinib in plasma.ResultsIn vitro analysis revealed that the IC50 value of osimertinib in rat liver microsomes was 27.6 μM. When rats were pretreated with avitinib, the values of AUC and MRT of the osimertinib were increased, and its Cmax and Tmax were significantly extended, whereas the values of CLz/F were significantly decreased (P < 0.05).ConclusionsBoth in vitro and in vivo results demonstrated that a drug‐drug interaction between avitinib and osimertinib occurred and more attention should be paid when avitinib and osimertinib are synchronously administered in clinic.Key pointsSignificant findings of the studyOsimertinib is the only market available third‐generation EGFR‐TKI and it has been reported that some drugs could have drug‐drug interactions with it.What this study addsFor the first time, we systematically investigated the effect of avitinib, one newly developed third‐generation EGFR‐TKI, on the pharmacokinetics of osimertinib both in vitro and in vivo using a rat model.

Context: Naringenin and tofacitinib are often used together for treatment of rheumatoid arthritis in Chinese clinics.Objective: This experiment investigates the effect of naringenin on the pharmacokinetics of tofacitinib in rats.Materials and methods: Twelve Sprague-Dawley rats were randomly divided into two groups (experimental group and control group). The experimental group was pre-treated with naringenin (150 mg/kg/day) for two weeks before dosing tofacitinib, and equal amounts of CMC-Na solution in the control group. After a single oral administration of 5 mg/kg of tofacitinib, 50 μL blood samples were directly collected into 1.5 mL heparinized tubes via the caudal vein at 0.083, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h. The plasma concentration of tofacitinib was quantified by UPLC/MS-MS.Results: Results indicated that naringenin could significantly affect the pharmacokinetics of tofacitinib. The AUC0-24 of tofacitinib was increased from 1222.81 ± 222.07 to 2016.27 ± 481.62 ng/mL/h, and the difference was significant (p < 0.05). Compared with the control group, the Tmax was increased from 0.75 ± 0.29 to 3.00 ± 0.00 h (p < 0.05), and the MRT(0-24) was increased from 4.90 ± 0.51 to 6.57 ± 0.66 h (p < 0.05), but the clearance was obviously decreased from 4.10 ± 0.72 to 2.42 ± 0.70 L/h/kg (p < 0.05) in experimental group. Although the Cmax and t1/2 of tofacitinib were increased, there were no significant differences (p > 0.05).Conclusions: This research demonstrated a drug-drug interaction between naringenin and tofacitinib possibly when preadministered with naringenin; thus, we should pay attention to this possibility in the clinic.

An ultra-liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of rivaroxaban, dabigatran, and clopidogrel in human plasma, using diazepam as an internal standard (IS). The samples were chromatographed on a CORTECS BEH C18 column (2.1 x 50 mm, 1.6 mu m) by a mobile phase consisting of methanol (0.1% formic acid) and water (0.1% formic acid) at a flow rate of 0.4 mL/min. The protonated ions of analytes were detected in positive ionization in multiple reaction monitoring mode (MRM). The mass transition pairs of m/z 435.93 -> 144.81, 472.47 -> 289.10, 322.16 -> 212.10, and 285.10 -> 193.10 were used to quantify rivaroxaban, dabigatran, clopidogrel and diazepam, respectively. The current method was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery, matrix effect and stability which proved to be satisfactory. The validated method was successfully applied to clinical therapeutic drug monitoring (TDM) in hospitalized patients.

An accurate and validated UPLC-MS/MS method for determination of folic acid and 5-methyltetrahydrofolic acid in human plasma were developed and validated. Plasma samples were prepared by precipitating protein with acetonitrile. The analytes were separated using a reversed-phase BEH C18 column (2.1 x 50 mm, 1.7 mu m, Waters, USA) maintained at 40 degrees C. The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) with a gradient elution pumped at a flow humane of 0.4 mL/min. The analytes were detected with positive electrospray ionization in multiple reaction monitoring (MRM) mode for target fragment ions m/z 442.11 -> 295.14 for folic acid, m/z 460.26 -> 313.23 for 5-methyltetrahydrofolic acid and m/z 285.1 -> 493.1 for diazepam (IS). Calibration plots were linear throughout the ranges of 1-500 ng/mL for folic acid and 0.25-100 ng/mL for 5-methyltetrahydrofolic acid in human plasma. The mean recoveries of folic acid and 5-methyltetrahydrofolic acid from the plasma exceeded 73.23%. The intra-run and inter-run assay precisions of folic acid and 5-methyltetrahydrofolic acid were both less than 9.77%. The validated method showed the capability of the method to be used as an alternative for plasma analysis in therapeutic drug monitoring.