Enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma and urine by liquid chromatography/tandem mass spectrometry

A sensitive method using enantiospecific liquid chromatography/tandem mass spectrometry detection for the quantitation of S- and R-mephenytoin as well as its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma and urine has been developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile, while urine samples were diluted twice with the mobile phase before injection. The analytes were then separated on a chiral alpha(1)-acid glycoprotein (AGP) column and thereafter detected, using electrospray ionization tandem mass spectrometry. In plasma, the lower limit of quantification (LLOQ) was 1 ng/mL for S- and R-4'-hydroxymephenytoin and S-nirvanol and 3 ng/mL for R-nirvanol and S- and R-mephenytoin. In urine, the LLOQ was 3 ng/mL for all compounds. Resulting plasma and urine intra-day precision values (CV) were <12.4% and <6.4%, respectively, while plasma and urine accuracy values were 87.2-108.3% and 98.9-104.8% of the nominal values, respectively. The method was validated for plasma in the concentration ranges 1-500 ng/mL for S- and R-4'-hydroxymephenytoin, 1-1000 ng/mL for S-nirvanol, and 3-1500 ng/mL for R-nirvanol and S- and R-mephenytoin. The validated concentration range in urine was 3-5000 ng/mL for all compounds. By using this method, the metabolic activities of two human drug-metabolizing enzymes, cytochrome P450 (CYP) 2C19 and CYP2B6, were simultaneously characterized.     

Sensitive and convenient method for the quantification of clonidine in serum of pediatric patients using liquid chromatography/tandem mass spectrometry

An improved and easy to use liquid chromatography/tandem mass spectrometric (LC/MS/MS) method in human serum was developed for the quantification of clonidine (CLD), an alpha2-/alpha1-adrenoceptor agonist, used for analgo-sedation and the therapy of opioid withdrawal in pediatric patients. Sample preparation consisted of precipitation of serum proteins by adding acetonitrile and centrifugation of the sample subsequently. [(2)H4]Clonidine (CLD4) served as internal standard. Chromatographic separation of the supernatant was achieved using a 100mmx3mm, 5microm Thermo Electron BetaBasic C4 column with isocratic flow and elution consisting of 0.1% formic acid/acetonitrile (85/15, v/v) and a flow-rate of 350microl/min resulting in a column pressure of 280-420kPa. LC/MS/MS detection was performed by using a triple-stage quadrupole mass spectrometer (TSQ Quantum, Thermo Electron) working in selected reaction monitoring mode with positive electrospray ionization. The analyte was quantified in a single run within 5min. Linearity was demonstrated over the expected concentration range 0.15-50microg/l CLD. The lower limit of quantification (LLOQ) and the limit of detection were 0.1microg/l and 0.01microg/l, respectively. None of the drugs used concomitantly during analgesic therapy interfered in the assay in vitro. Intra-day precision expressed as RSD was 9.6% or less for CLD, while inter-day result was 10.0% or less for CLD. Intra-day and inter-day accuracy was within +/-4.9% and +/-1.8%, respectively. The method was validated according to the international guidelines of the International Conference on Harmonisation (ICH) and the US Food and Drugs Administration (FDA). The described method is suitable to analyse serum samples with very small volumes and sets the stage for pharmacokinetic studies in pediatric studies.     

Determination of HX0969w in rabbit whole blood by liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study

HX0969w is a novel carboxylate ester prodrug of propofol. After intravenous administration of HX0969w, the compound is rapidly hydrolyzed to its active metabolite propofol. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with electrospray ionization has been developed for determination of HX0969w in rabbit whole blood. Protein precipitation with methanol was used for sample preparation and 7-hydroxycoumarin served as internal standard. The standard curve ranged from 50.1 to 15,030 ng mL(-1). The lower limit of quantification (LLOQ) for HX0969w was 50.1 ng mL(-1). The intra- and inter-day accuracies were within ± 10% and precisions were below 5.52%. The method was successfully applied to samples from a rabbit pharmacokinetic study.     

The Development of New Methodology for Determination of Vincristine (VCR) in Human Serum Using LC-MS/MS-Based Method for Medical Diagnostics

In this article, we have presented the development and validation of a rapid and sensitive reversed-phase liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of vincristine (VCR) in patient serum samples. Chromatographic separation was achieved on a Kinetex^® (Singapore) column using a mobile phase consisting of 25 mM acetic acid and 0.3% formic acid (A) and methanol (B) in a gradient elution mode at a flow rate of 0.3 mL/min. The VCR and internal standard (vinblastine) were monitored using the multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantification (LLOQ) was 0.67 ng/mL, and the upper limit of quantification (ULOQ) was 250 ng/mL for VCR. The calculated values of LOD and LOQ for VCR were 0.075 and 0.228 ng/mL, respectively. The calibration curve was linear over the VCR concentration range of 1.0–250 ng/mL in serum. The intra- and inter-day precision and precision were within the generally accepted criteria for the bioanalytical method (<15%). The method was successfully applied to the analysis of serum samples in clinical practice.     

Development of a liquid chromatography/electrospray tandem mass spectrometry assay for the quantification of apicidin, a novel histone deacetylase inhibitor, in rat serum: application to a pharmacokinetic study

Apicidin, a fungal metabolite isolated from Fusarium pallidoroseum, is a cyclic tetrapeptide that exhibits potent anti-protozoal and anti-angiogenic activities. Although extensive studies have been recently conducted to examine the biological and pharmacological action, no information is available on the quantitative analysis of apicidin. To our knowledge, this study is the first to describe a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay method for the quantification of apicidin in rat serum. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), accuracy, and precision. The multiple reaction monitoring was based on the transitions m/z 624.7 --> 84.3 and 372.1 --> 176.1 for apicidin and trazodone, respectively. The assay utilized a single liquid-liquid extraction and isocratic elution, and the LLOQ was 0.5 ng/mL using 0.1 mL of rat serum. The assay was linear over a range from 0.5-1000 ng/mL, with correlation coefficients >0.9994. The mean intra- and inter-day assay accuracy ranged from 99.9-101.5% and 94.8-102.1%, respectively, and the mean intra- and inter-day precision was between 2.7-5.9% and 1.6-11.5%, respectively. The developed assay method was applied to a pharmacokinetic study after intravenous injection of apicidin in rats at a dose of 1 mg/kg.     

LC-MS/MS法快速测定吡罗昔康制剂中吡罗昔康含量

建立了快速液相色谱-质谱/质谱联用法测定吡罗昔康制剂中吡罗昔康含量的方法。样品以0.1 mol/L盐酸甲醇溶液提取、微孔滤膜过滤、离心后,通过电喷雾离子化(ESI),采用多反应检测(MRM)方式进行正离子检测,用于定量分析的检测离子为m/z 332.2→94.8。采用Shim-pack XR-ODS(3.0 mm×75mm,2.0μm)柱分离,以乙腈-水-甲酸(60:40:0.1,V/V/V)为流动相,流速为0.40 mL/min,在3 min内完成吡罗昔康定量分析。线性范围为2.5～1000.0ng/mL,最低检测限为2.5 ng/mL;日内测定的相对标准偏差小于3.2%,日间测定的相对标准偏差小于3.8%。方法可作为吡罗昔康制剂的质量中吡罗昔康控制方法,并可用于少量血浆样品的测定,也适用于药物代谢动力学研究。     

Liquid chromatography/mass spectrometric bioanalysis of a modified gamma-cyclodextrin (Org 25969) and Rocuronium bromide (Org 9426) in guinea pig plasma and urine: its application to determine the plasma pharmacokinetics of Org 25969

A sensitive and specific liquid chromatography/mass spectrometry (LC/MS) method has been developed and validated for the quantification of the modified gamma-cyclodextrin Org 25969 and Rocuronium bromide (Roc or Org 9426) in the plasma and urine of guinea pigs. The assay was linear and reproducible over the range 25-10000 ng/mL for both compounds. The lowest limit of quantification (LLOQ) for both compounds in urine was 25 ng/mL. In plasma, the LLOQ was 25 ng/mL for Org 9426 and 50 ng/mL for Org 25969. The inter- and intra-day variation was lower than 20%. The physicochemical properties of both compounds imposed different modes of extraction from plasma. The modified gamma-cyclodextrin was extracted by trifluoroacetic acid (TFA) precipitation while Rocuronium was extracted by acetonitrile precipitation. Both compounds were quantified in urine by direct injection onto the column. The LC/MS analyses of Org 25969 and Org 9426 were performed using two different assay conditions. It was not possible to quantify the complex of cyclodextrin and Roc as it dissociated on the LC column. The use of LC/MS conferred great advantage to the quantification of both Org 25969 and Org 9426, as they were not chromogenic enough to afford the sensitivity and specificity required for the assay.     

A UPLC-MS/MS assay of the "Pittsburgh cocktail": six CYP probe-drug/metabolites from human plasma and urine using stable isotope dilution

The efficiency of drug metabolism by a single enzyme can be measured as the fractional metabolic clearance which can be used as a measure of whole body activity for that enzyme. Measurement of activity of multiple enzymes simultaneously is feasible using a cocktail approach, however, analytical approach using different assays for drug probes can be cumbersome. A quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based method for the rapid measurement of six cytochrome P450 (CYP) probe drugs and their relevant metabolites is described. The six specific probe substrates/metabolites are caffeine/paraxanthine (CYP1A2), flurbiprofen/4'-hydroxyflurbiprofen (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), debrisoquine/4-hydroxydebrisoquine (CYP2D6), chlorzoxazone/6'-hydroxychlorzoxazone (CYP2E1) and dapsone/N-monoacetyldapsone (NAT2). These probes were quantified by stable isotope dilution from plasma and urine. The present workflow provides a robust, fast and sensitive assay for the "Pittsburgh cocktail", and has been successfully applied to a clinical phenotyping study of liver disease. A representative group of 17 controls and patients with chronic liver disease were administered orally caffeine (100 mg), chlorzoxazone (250 mg), debrisoquine (10 mg), mephenytoin (100 mg), flurbiprofen (50 mg) and dapsone (100 mg). Urine (0 through 8 h) and plasma (4 and 8 h) samples were analyzed for drug/metabolite amounts by stable isotope dilution UPLC-MS/MS. The phenotypic activity of drug metabolizing enzymes was investigated with 17 patient samples. Selected reaction monitoring (SRM) was optimized for each drug and metabolite. In the method developed, analytes were resolved by reversed-phase by development of a gradient using a water/methanol solvent system. SRM of each analyte was performed in duplicate on a triple quadrupole mass spectrometer utilizing an 8 min analytical method each, one with the source operating in the positive mode and one in the negative mode, using the same solvent system. This method enabled quantification of each drug (caffeine, chlorzoxazone, debrisoquine, mephenytoin, flurbiprofen, and dapsone) and its resulting primary metabolite in urine or plasma in patient samples. The method developed and the data herein demonstrate a robust quantitative assay to examine changes in CYP enzymes both independently or as part of a cocktail. The clinical use of a combination of probe drugs with UPLC-MS/MS is a highly efficient tool for the assessment of CYP enzyme activity in liver disease.     

Determination of clopidogrel in human plasma by liquid chromatography/tandem mass spectrometry: application to a clinical pharmacokinetic study

A rapid and sensitive LC/MS/MS assay was developed and validated for the determination of clopidogrel in human plasma. Clopidogrel was extracted by single liquid-liquid extraction with pentane, and chromatographic separations were achieved on a C(18) column. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), stability, accuracy and precision. The multiple reaction monitoring was based on m/z transition of 322.2 --> 211.9 for clopidogrel and 264.1 --> 125.1 for ticlopidine (internal standard). The total analytical run time was relatively short (3 min), and the LLOQ was 10 pg/mL using 0.5 mL of human plasma. The assay was linear over a concentration range from 10 to 10,000 pg/mL (r > 0.999). The intra- and inter-day accuracies were 101.3-108.8 and 98.4-103.5%, respectively, and the intra- and inter-day assay precisions were 1.9-5.5 and 4.4-8.1%, respectively. The developed assay method was applied to a pharmacokinetic study in human volunteers after oral administration of clopidogrel at a dose of 150 mg.     

Quantitative determination of cetirizine enantiomers in guinea pig plasma, brain tissue and microdialysis samples using liquid chromatography/tandem mass spectrometry

Sensitive enantioselective liquid chromatographic assays using tandem mass spectrometric detection were developed and validated for the determination of S-cetirizine (S-CZE) and R-cetirizine (R-CZE) in guinea pig plasma, brain tissue, and microdialysis samples. Enantioselective separation was achieved on an alpha1-acid glycoprotein column within 14 min for all methods. A cetirizine analog, ucb 20028, was used as internal standard. Cetirizine and the internal standard were detected by multiple reaction monitoring using transitions m/z 389.1 --> 200.9 and 396.1 --> 276.1, respectively. The samples were prepared using protein precipitation with acetonitrile. For guinea pig plasma, the assay was linear over the range 0.25-5000 ng/mL for both S-CZE and R-CZE, with a lower limit of quantification (LLOQ) of 0.25 ng/mL. For the brain tissue and microdialysis samples, the assays were linear over the range 2.5-250 ng/g and 0.25-50 ng/mL, respectively, and the LLOQ values were 2.5 ng/g and 0.25 ng/mL, respectively. The intra- and inter-day precision values were < or =7.1% and < or =12.6%, respectively, and the intra- and inter-day accuracy varied by less than +/-8.0% and +/-6.0% of the nominal value, respectively, for both enantiomers in all the matrices investigated.     

Determination of tubuloside B by LC‐MS/MS and its application to a pharmacokinetic study in rats

Tubuloside B, a novel neuroprotective phenylethanoid, is a major active constituent of Cistanche tubulosa and Cistanche deserticola. A specific and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for the quantification of tubuloside B in rat plasma. Sample preparation was conducted through a protein‐precipitation extraction with methanol using tubuloside A as internal standard (IS). Chromatographic separation was achieved using a Capcell Pak C₁₈ column (2.0 × 50 mm, 5 μm) with a mobile phase of methanol–10 mm ammonium acetate buffer (70:30, v/v) in an isocratic elution. Mass spectrometry analysis was performed in negative ionization mode with selected reaction monitoring transitions at m/z 665.1 → 160.9 for tubuloside B, and m/z 827.1 → 160.9 for IS. Calibration curves were linear over the range of 1.64–1640 ng/mL for plasma samples samples (R² > 0.990). The lower limit of quantification (LLOQ) was 1.64 ng/mL. The intra‐ and inter‐day accuracy was between 92.3 and 113.0% with the RSD <9.23% at all LLOQ and quality control levels. Finally, this method was successfully applied in the pharmacokinetics study of tubuloside B after intravenous administration.     

A simple RP-HPLC method for the simultaneous determination of curcumin and its prodrug, curcumin didecanoate, in rat plasma and the application to pharmacokinetic study

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of curcumin and its prodrug, curcumin didecanoate (CurDD), in rat plasma. The analytes were extracted by ethyl acetate following the addition of sodium dodecyl sulfate, and separated on a reverse-phase C(18) column using a gradient mobile phase system of acetonitrile-tetrahydrofuran-water containing 0.1% formic acid. Detection by UV absorption at 425 nm gave a lower limit of quantitation (LLOQ) of 5 and 10 ng/mL for curcumin and CurDD in 50 μL of plasma, respectively. Intra- and inter-day precisions of quality control samples except those at LLOQ were within 15% for curcumin and CurDD, respectively, and the accuracies for both compounds were between 93.9 and 108%. The method was successfully applied to determine plasma concentration-time curves of curcumin and CurDD in rats following intravenous (i.v.) administration of curcumin or CurDD at doses of 1 mg/kg (calculated as curcumin). The results suggested that i.v. dosed CurDD provided sustained plasma levels of curcumin.     

HPLC method for determination of diclofenac in human plasma and its application to a pharmacokinetic study in Turkey

A simple high-performance liquid chromatography (HPLC) method has been developed for determination of diclofenac in human plasma. The method was validated on Ace C(18) column using UV detection. The mobile phase consisted of 20 mM phosphate buffer (pH 7) containing 0.1% trifluoroacetic acid-acetonitrile (65:35, v/v). Calibration curve was linear between the concentration range of 75-4000 ng/mL. Intra- and inter-day precision values for diclofenac in plasma were less than 3.6, and accuracy (relative error) was better than 5.3%. The limits of detection and quantification of diclofenac were 25 and 75 ng/mL, respectively. Also, this assay was applied to determine the pharmacokinetic parameters of diclofenac in healthy Turkish volunteers who had been given 50 mg diclofenac.     

Determination of the lipophilic antipsychotic drug ziprasidone in rat plasma and brain tissue using liquid chromatography-tandem mass spectrometry

A simple, sensitive and robust liquid chromatography/electrospray ionization tandem mass spectrometry (LCESI-MS/MS) method with low matrix effects was developed and validated for the quantification of the lipophilic antipsychotic ziprasidone from rat plasma and brain tissue. Ziprasidone was extracted from rat plasma and brain homogenate using a single-step liquid-liquid extraction. Ziprasidone was separated on an Agilent Eclipse XDB C8 column (150 x 2.1 mm i.d., 5 microm) column using a mobile phase of acetonitrile-0.02% ammonia in water (pH 7.20 adjusted with formic acid) using gradient elution. Ziprasidone was detected in the positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, lower limit of quantitation (LLOQ), precision, accuracy, recovery, matrix effects and stability were determined. The LLOQ was 0.2 ng/mL for plasma and 0.833 ng/g for brain tissue. The method was linear over the concentration range from 0.2 to 200.0 ng/mL for plasma and 0.833-833.3 ng/g for brain tissue. The correlation coefficient (R2) values were more than 0.996 for both plasma and brain homogenate. The precision and accuracy intra-day and inter-day were better than 8.13%. The relative and absolute recovery was above 81.0% and matrix effects were lower than 5.2%. This validated method has been successfully used to quantify the rat plasma and brain tissue concentration of ziprasidone after chronic treatment.     

Determination of midazolam and its hydroxy metabolites in human plasma and oral fluid by liquid chromatography/electrospray ionization ion trap tandem mass spectrometry

Midazolam (MDZ), a short-acting benzodiazepine, is a widely accepted probe drug for CYP3A phenotyping. Published methods for its analysis have used either therapeutic doses of MDZ, or, if employing lower doses, were mostly unable to quantify the two hydroxy metabolites. In the present study, a sensitive and specific liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the quantitative determination of MDZ and two of its metabolites (1'-hydroxymidazolam (1'-OHMDZ) and 4-hydroxymidazolam (4-OHMDZ)) in human plasma and oral fluid. After liquid-liquid extraction with hexane/dichloromethane (73:27, v/v), the analytes were separated on a Luna C18(2) (100 x 2.1 mm) analytical column using gradient elution. Detection was achieved using tandem mass spectrometry on an ion trap mass spectrometer. Midazolam-d6 was used as internal standard for quantification. The calibration curves were linear (R2 >0.998) between 0.05 and 20 ng/mL for MDZ and both metabolites in both matrices. Using 1 mL samples, the limit of detection was 0.025 ng/mL and the limit of quantification was 0.05 ng/mL for MDZ and the hydroxy metabolites in both matrices. Intra- and inter-day accuracies, determined at three different concentrations, were between 92.1 and 102.3% and the corresponding coefficients of variation were <7.3%. The average recoveries were 90.6%, 86.7% and 79.0% for MDZ, 1'-OHMDZ and 4-OHMDZ in plasma and 95.3%, 96.6% and 86.8% for MDZ, 1'-OHMDZ and 4-OHMDZ, respectively, in oral fluid. The method was successfully applied to a pharmacokinetic study, showing that MDZ and its hydroxy metabolites can be determined precisely in in vivo samples obtained following a single oral or intravenous dose of 2 mg MDZ. The method appears to be useful for CYP3A phenotyping in plasma using sub-therapeutic MDZ doses, but larger studies are needed to test this assumption.     

A rapid and specific approach for direct measurement of donepezil concentration in human plasma by LC-MS/MS employing solid-phase extraction

A selective, rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for assay of donepezil in human plasma using escitalopram as an internal standard. Chromatographic separation was achieved on a Betabasic-C(8), 5 microm, 100 x 4.6 mm column using methanol:water:formic acid (90:9.97:0.03, v/v/v) as mobile phase. Detection of donepezil and internal standard was achieved by ESI MS/MS in positive ion mode using 380.20/91.10 and 325.13/262.00 transitions, respectively. The linearity over the concentration range of 0.15-50 ng/mL for donepezil was obtained and the lower limit of quantification was 0.15 ng/mL. For each level of quality control samples, inter-day and intra-day precisions (RSD) were < or =8.92 and 10.35% and accuracy (%RE) were < or =7.33% and 9.33%, respectively. The recovery was more than 88.50% for both donepezil and internal standard by solid-phase extraction, eliminating evaporation and reconstitution steps.     

Development and validation of a liquid chromatographic/tandem mass spectrometric method for the determination of sertraline in human plasma

A sensitive and rapid liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of sertraline in human plasma. The analyte and internal standard (IS, diphenhydramine) were extracted with 3 mL of diethyl ether/dichloromethane (2:1, v/v) from 0.25 mL plasma, then separated on a Zorbax Eclipse XDB C18 column using methanol/water/formic acid (75:25:0.1, v/v/v) as the mobile phase. The triple quadrupole mass spectrometry was applied via an atmospheric pressure chemical ionization (APCI) source for detection. The fragmentation pattern of the protonated sertraline was elucidated with the aid of product mass spectra of isotopologous peaks. Quantification was performed using selected reaction monitoring of the transitions of m/z 306 --> 159 for sertraline and m/z 256 --> 167 for the IS. The method was linear over the concentration range of 0.10-100 ng/mL. The intra-day and inter-day precisions, expressed by relative standard deviation, were both less than 6.7%. Assay accuracies were within +/-6.9% as terms of relative error. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.10 ng/mL with a precision of 8.3% and an accuracy of 9.6%. The validated method has been successfully applied for the pharmacokinetic study and bioequivalence evaluation of sertraline in 18 healthy volunteers after a single oral administration of 50 mg sertraline hydrochloride tablets.     

基质分散固相萃取-高效液相色谱/串联质谱法检测红薯中氯吡脲

建立了分散固相萃取-高效液相色谱/串联质谱法测定红薯中氯吡脲含量的分析方法.样品经分散剂(硅胶,C18HC)研磨分散提取,甲醇洗脱.采用Thermo Hypersil Gold C18色谱柱(150×2,1 mm,5μm)分离,以甲醇(A)/0.1％甲酸-5 mmol/L甲酸铵(B)作为流动相,梯度洗脱,采用串联质谱在正离子扫描方式下,通过选择反应模式定量分析,外标法定量.氯吡脲在2.63～675.00 ng/mL范围内线性关系良好,相关系数R2＞0.999.定量检测限为0.66 ng/mL.氯吡脲在10、25、50 ng/g三个浓度水平的添加回收率在70％～130％之间.该方法操作简单、准确,适用于红薯中氯吡脲的定量检测.     

Simultaneous determination of the bioactive components in rat plasma by UPLC‐MS/MS and application in pharmacokinetic studies after oral administration of radix Scutellariae extract

A highly sensitive and rapid ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantification of the four main bioactive compounds, i.e. baicalin, baicalein, wogonoside and wogonin, in rat plasma after oral administration of Radix Scutellariae extract. Clarithromycin was used as an internal standard (IS). Plasma samples were processed by protein precipitation with methanol. The separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm, 1.7 μm) at a flow rate of 0.4 mL/min, using 0.1% formic acid–acetonitrile as mobile phase. The MS/MS ion transit ions monitored were 447.5 → 270.1 for baicalin, 270.1 → 168.1 for baicalein, 461.2 → 284.0 for wogonoside, 284.2 → 168.1 for wogonin and 748.5 → 158.1 for IS. Method validation was performed according to US Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantification (LLOQ) achieved was 1.13 ng/mL for baicalin, 1.23 ng/mL for baicalein, 0.82 ng/mL for wogonoside and 0.36 ng/mL for wogonin. The calibration curves obtained were linear (r > 0.99) over the concentration range ~ 1–1000 ng/mL. The intra‐ and inter‐day precision was <15% and the accuracy was within ±14.7%. After validation, this method was successfully applied to a pharmacokinetic study of Radix Scutellariae extract.     

Rapid and sensitive LC-MS/MS method for the determination of metoprolol in Beagle dog plasma with a simple protein precipitation treatment and its pharmacokinetic applications

A rapid LC-MS/MS method with good accuracy and sensitivity was developed and validated for the pharmacokinetics study of metoprolol (MP) in beagle dogs. The plasma samples were simply precipitated by methanol and then analyzed by LC-MS/MS. An Ultimate XB-C?? column (150 × 2.1 mm ID, 5 μm) was used for separation, with methanol-water containing 0.2% formic acid (65:35, v/v) as the mobile phase at a flow rate of 0.2 mL/min. Monitoring ions of MP and internal standard (hydroxypioglitazone) were m/z 268.1/115.6 and m/z 373.1/150.2, respectively. The linear range was 3.03-416.35 ng/mL with an average correlation coefficient of 0.9996, and the limit of quantification was 3.03 ng/mL. The intra- and inter-day precision was less than 15%. At low, middle and high concentrations, the recovery, the matrix effect and the accuracy was in the range of 76.06%-95.25%, 93.67%-104.19% and 95.20%-99.96% respectively. The method was applied for the pharmacokinetics study of MP tartrate tablets (50 mg). The AUC(0-t), T(max) and C(max) were respectively 919.88 ± 195.67 μg/L·h, 0.96 ± 0.33 h, 349.12 ± 78.04 ng/mL.