Liquid chromatographic tandem mass spectrometry method for the quantification of miglitol in human plasma

A rapid, sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of miglitol (CAS 72432-03-2), an alpha-glucosidase inhibitor, in human plasma using gabapentin (CAS 60142-96-3) as internal standard (IS). Following protein precipitation, the analytes were separated using an isocratic mobile phase on a reversed phase phenyl column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 208/146 for miglitol and m/z 172/154 for the IS. The assay exhibited a linear dynamic range of 100-6000 ng/mL for miglitol in human plasma. The lower limit of quantification was 100 ng/mL with a relative standard deviation of less than 5 %. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. The average absolute recoveries of miglitol and the IS from spiked plasma samples were 40.5 +/- 2.7 and 47.1 +/- 2.9 %, respectively. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The miglitol plasma concentration profile could be obtained for pharmacokinetic study. The observed maximum plasma concentration (Cmax) of miglitol (100 mg oral dose) is 1740 ng/mL, time to observed maximum plasma concentration (tmax) is 3.5 h and elimination half-life (t(1/2)) is 2.5 h.     

Liquid chromatography-negative ion electrospray tandem mass spectrometry method for the quantification of ezetimibe in human plasma

A simple, reliable and sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for quantification of free and total ezetimibe in human plasma. The analyte and internal standard (13C6-ezetimibe) were extracted by liquid-liquid extraction with methyl tert-butyl ether. The reversed-phase chromatographic separation was performed on a Capcell C18 column, and the plasma extract was eluted with a gradient consisting of acetonitrile and 5 mM ammonium acetate. The analyte was detected using negative ionization by multiple reaction monitoring mode. The mass transition pairs of m/z 408.5-->270.8 and m/z 414.5-->276.8 were used to detect ezetimibe and internal standard, respectively. The assay exhibited linear ranges from 0.02 to 20 ng/ml for free ezetimibe and 0.25 to 250 ng/ml for total ezetimibe in human plasma. Acceptable precision and accuracy were obtained for concentrations of the calibration standard and quality control. The validated method was successfully used to analyze human plasma samples for application in a pharmacokinetic study.     

Determination of pinostrobin in rat plasma by LC-MS/MS: application to pharmacokinetics

A rapid and sensitive method for the determination of pinostrobin in rat plasma was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the first time. Isoliquiritigenin was used as an internal standard in rat plasma. Chromatographic separation was performed on an HiQ Sil C₁₈ column with isocratic elution at a flow rate of 1 mL/min. The mobile phase consisted of water and methanol (9:91, v/v) containing 0.1% formic acid. The quantification limit was 10 ng/mL within a linear range of 10-1000 ng/mL (R = 0.9984). The intra- and inter-day assay precision ranged from 3.8-5.3% to 3.2-5.2%, respectively, and the intra- and inter-day assay accuracy was between 93.2-95.1% and 95.5-104.3%, respectively. Our results indicated that the LC-MS/MS method is effective for pharmacokinetic study of pinostrobin in rat plasma.     

Rapid quantification of gabapentin in human plasma by liquid chromatography/tandem mass spectrometry

A simple, sensitive and rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of gabapentin, a new antiepileptic drug, in human plasma using its structural analogue, 1,1-cyclohexane diacetic acid monoamide (CAM) as internal standard. The method involved a simple protein precipitation by means of acetonitrile followed by a rapid isocratic elution with 10mM ammonium formate buffer/acetonitrile (20/80, v/v, pH 3.0) on Waters Symmetry C(18 reversed phase chromatographic column and analyzed by mass spectrometry in the multiple reaction monitoring mode. The precursor to product ion transitions of m/z 172-->154 and m/z 200-->182 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 40-10000 ng/mL for gabapentin in human plasma. The limit of detection and lower limit of quantification in human plasma were 10 and 40 ng/mL, respectively. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

LC-MS/MS法测定人血浆中加巴喷丁的浓度及其药动学研究

建立液相色谱串联质谱(LC-MS/MS)法测定人血浆中加巴喷丁的浓度并将其应用于人体药动学研究。取血浆样品经甲醇沉淀蛋白后,以甲醇0.2%甲酸水溶液(80∶20)为流动相,用Inertsil ODS-3 C18柱(50 mm×2.1 mm ID,3μm)分离,采用电喷雾离子源,以多反应监测(MRM)方式进行正离子检测,定量分析的离子反应分别为m/z 172→m/z 154(加巴喷丁)和m/z 130→m/z 71(内标二甲双胍)。加巴喷丁线性范围为40.8～8.16×103 ng.mL 1,定量限为40.8 ng.mL 1,每个样品测试时间仅2.2 min,日内、日间精密度(RSD)均小于12%,准确度(RE)在±6.4%范围内。应用此法研究了20名健康志愿者单剂量口服加巴喷丁胶囊600 mg后的药动学特点。该方法快速、专属、灵敏、适用性强,可应用于加巴喷丁的人体药动学研究。     

High performance liquid chromatography-tandem mass spectrometric determination of rupatadine in human plasma and its pharmacokinetics

A simple, rapid, sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of rupatadine in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-ammonium acetate (pH 2.2; 5mM) (50:50, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the positive ion and multiple reaction monitoring (MRM) mode, m/z 416-->309 for rupatadine and m/z 295-->267 for the IS. The assay exhibited a linear dynamic range of 0.1-100 ng/ml for rupatadine in human plasma. The lower limit of quantification (LLOQ) was 0.1 ng/ml with a relative standard deviation of less than 20%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of rupatadine in healthy volunteers.     

Development and validation of a high-throughput assay for quantification of the proliferation inhibitor ABT-578 using LC/LC-MS/MS in blood and tissue samples

We report here a specific, automated LC/LC-MS/MS assay for the quantification of ABT-578 in human and rabbit blood and rabbit tissues for drug-eluting stent development. After protein precipitation, samples were injected into the HPLC system and extracted online using a high flow of 5 mL/min. The extracts were then backflushed onto the analytic column. The [M+Na] of ABT-578 (m/z 988.6-->369.4) and its internal standard sirolimus (m/z 936.5-->409.3) were monitored. Extraction and analysis took 4 minutes. The assay was validated following the US Food & Drug Administration guidelines. Linearity was 0.025-25 ng/mL for most matrices. In human blood, interday accuracies were 81.8% (at 0.025 ng/mL), 91.0% (1 ng/mL), and 99.5% (50 ng/mL), and interday precisions were 10.7% (0.025 ng/mL), 3.0% (1 ng/mL), and 1.8% (50 ng/mL).     

Rapid and simultaneous measurement of midazolam, 1'-hydroxymidazolam and digoxin by liquid chromatography/tandem mass spectrometry: application to an in vivo study to simultaneously measure P-glycoprotein and cytochrome P450 3A activity

In order to simultaneously determine in vivo P-glycoprotein (P-gp) and Cytochrome P450 3A (CYP3A) activity, a new, rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to simultaneously determine midazolam (MDZ, as CYP3A substrate), 1'-hydroxymidazolam (1'-OHMDZ) and digoxin (DG, as P-gp substrate) in rat plasma using digitoxin as the internal standard (IS). After a single step liquid-liquid extraction with tert-butyl methyl ether/dichloromethane (75:25, v/v), analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). Chromatographic separation was performed on an XTerra MS C18 column (50mm×2.1mm, i.d. 3.5μm). The MS/MS detection was conducted by monitoring the fragmentation of 326.05 → 244.00 (m/z) for MDZ, 342.02 →168.01 (m/z) for 1'-OHMDZ, 798.33 → 651.36(m/z) for DG and 782.67 → 635.24 (m/z) for IS. The method had a chromatographic running time of 3min and linear calibration curves over the concentrations of 2-400ng/mL for MDZ and 1'-OHMDZ and 0.5-100ng/mL for DG. The recoveries of the method were 86.8-96.3% for MDZ, 84.6-86.4% for 1'-OH MDZ, and 81.7-85.1% for DG. The lower limit of quantification (LLOQ) of the method was 2ng/mL for MDZ and 1'-OHMDZ and 0.5ng/mL for DG. The intra- and inter-batch precision were less than 15% for all quality control samples at concentrations of 5, 50 and 320ng/mL for MDZ and 1'-OHMDZ and 1, 10 and 80ng/mL for DG. The validated LC-MS/MS method has been successfully used to analyze the concentrations of MDZ, 1'-OH MDZ and DG in rat plasma for simultaneous measurement of in vivo P-gp and CYP 3A activity.     

Determination of zabofloxacin in rat plasma by liquid chromatography with mass spectrometry and its application to pharmacokinetic study

The objective of the present study was to develop a rapid and sensitive method for the determination of zabofloxacin, a novel, broad-spectrum fluoroquinolone antibiotic, in rat plasma. Rat plasma samples were deproteinized with methanol, and then were injected into an LC-MS system for quantification. Zabofloxacin and enrofloxacin, which served as an internal standard, were analyzed by selected ion monitoring (SIM) at m/z transitions of 402 for zabofloxacin and 360 for the internal standard. The lower limit of quantification (LLOQ) was determined to be 10 ng/mL, with acceptable linearity ranging from 10 to 5000 ng/mL (R>0.999). The validation parameters for zabofloxacin, such as absolute matrix effect (107.7-116.0%), accuracy (92.5-101.1% for intra-day and 90.3-103.8% for inter-day), precision (7.7-10.2% for intra-day and 4.2-8.9% for inter-day), and stability in rat plasma (96.0-101.8%), were found to be acceptable according to the assay validation guidelines of the FDA (2001). Following oral administration of zabofloxacin to rats at a dose of 20mg/kg, the concentration of zabofloxacin in plasma was quantifiable in plasma samples collected up to 8h following zabofloxacin administration. The method described in the present study is applicable to routine pharmacokinetic studies in rats.     

Sensitive quantification of apomorphine in human plasma using a LC-ESI-MS-MS method

An analytical method based on liquid chromatography coupled with ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the identification and quantification of apomorphine in human plasma. Apomorphine was isolated from 0.5 mL of plasma using a liquid-liquid extraction with diethyl ether and boldine as internal standard, with satisfactory extraction recoveries. Analytes were separated on a 5-microm C18 Highpurity (Thermohypersil) column (150 mm x 2.1 mm I.D.) maintained at 30 degrees C, coupled to a precolumn (C18, 5-microm, 10 mm x 2.0 mm I.D., Thermo). The elution was achieved isocratically with a mobile phase of 2 mM NH4COOH buffer pH 3.8/acetonitrile (50/50, vol/vol) at a flow rate of 200 microL per minute. Data were collected either in full-scan MS mode at m/z 150 to 500 or in full-scan tandem mass spectrometry mode, selecting the [M+H]ion at m/z 268.0 for apomorphine and m/z 328.0 for boldine. The most intense daughter ion of apomorphine (m/z 237.1) and boldine (m/z 297.0) were used for quantification. Retention times were 2.03 and 2.11 minutes for boldine and apomorphine, respectively. Calibration curves were linear in the 0.025 to 20 ng/mL range. The limits of detection and quantification were 0.010 ng/mL and 0.025 ng/mL, respectively. Accuracy and precision of the assay were measured by analyzing 54 quality control samples for 3 days. At concentrations of 0.075, 1.5, and 15 ng/mL, intraday precisions were less than 10.1%, 5.3%, and 3.8%, and interday precisions were less than 4.8%, 6.6%, and 6.5%, respectively. Accuracies were in the 99.5 to 104.2% range. An example of a patient who was given 6 mg of apomorphine subcutaneously is shown, with concentrations of 14.1 ng/mL after 30 minutes and 0.20 ng/mL after 6 hours. The method described enables the unambiguous identification and quantification of apomorphine with very good sensitivity using only 0.5 mL of sample, and is very convenient for therapeutic drug monitoring and pharmacokinetic studies.     

Analysis of topiramate and its metabolites in plasma and urine of healthy subjects and patients with epilepsy by use of a novel liquid chromatography-mass spectrometry assay

A novel liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for quantification of topiramate (TPM) and its metabolites 10-hydroxy topiramate (10-OH-TPM), 9-hydroxy topiramate (9-OH-TPM), and 4,5-O-desisopropylidene topiramate (4,5-diol-TPM) in plasma and urine. The method uses 0.5 mL of plasma or 1 mL of urine that is extracted with diethyl ether and analyzed by LC-MS. Positive ion mode detection enables tandem mass spectrometric (MS/MS) identification of the aforementioned four compounds. Calibration curves of TPM, 4,5-diol-TPM, 9-OH-TPM, and 10-OH-TPM in plasma and urine were prepared and validated over the concentration range of 0.625 to 40 microg/mL using TPM-d(12) as an internal standard. Calibration curves were linear over this concentration range for TPM and its metabolites. Accuracy and precision ranged in urine from 83% to 114% and 4% to 13% (%CV), respectively, and in plasma from 82% to 108% and 6% to 13%, respectively. The applicability of the assay was evaluated by analyzing plasma samples from a healthy subject who received a single oral dose of TPM (200 mg) and urine samples from 11 patients with epilepsy treated with TPM (daily dose between 100 to 600 mg) alone or with other antiepileptic drugs. Only TPM was detected and quantified in the plasma samples, and its concentration ranged between 0.7 and 4.3 microg/mL. The concentrations of TPM and 10-OH TPM were quantifiable in all urine samples and ranged from 20 to 300 microg/mL for TPM and from 1 to 50 microg/mL for 10-OH-TPM. The metabolites 4,5-diol-TPM and 9-OH-TPM were also detected in all urine samples, but their concentrations were quantifiable only in 4 patients. An unidentified peak in the chromatograms obtained from patients' urine was attributed to 2,3-O-desisopropylidene topiramate (2,3-diol-TPM). Due to a lack of reference material of 2,3-diol TPM and the similar MS/MS spectrum with 4,5-diol-TPM, the calibration curves of 4,5-diol-TPM were used for the quantification of its isomer 2,3-diol-TPM. Based on these determinations, the apparent 2,3-diol-TPM-to-TPM concentration ratio in patients' urine ranged from 0.05 to 0.51 and the 10-OH-TPM-to-TPM ratio ranged from 0.02 to 0.17. In conclusion, a novel LC-MS method for the assay of TPM and four of its metabolites in plasma and urine was developed. Its utilization for analysis of urine samples from patients with epilepsy showed that the method was suitable for analysis of TPM and its metabolites in clinical samples. Two quantitatively significant TPM metabolites (10-OH-TPM and 2,3-diol-TPM) and two quantitatively minor metabolites (9-OH-TPM and 4,5-diol-TPM) were detected and quantified in urine samples from patients with epilepsy.     

Development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of sunitinib in rabbit plasma

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the measurement of sunitinib in rabbit plasma. After protein precipitation with acetonitrile, samples were analyzed on a Zorbax Extend-C₁₈ column (150 mm×4.6 mm, 5μm). The mobile phase consisted of a mixture of acetonitrile and deionized water (containing 0.05% formic acid)at a ratio of 27:73 (v/v), and the flow rate was set at 0.8 mL/min.The column temperature was maintained at 30 ^oC. The LC eluate was detected by an electrospray ionization (ESI) source operated in the positive ion mode, and quantification was conducted using MRM of the transitions m/z 399.24→283.01 and m/z 415.19→178.00 for sunitinib and internal standard (IS, diltiazem hydrochloride), respectively. The calibration curve was linear in the range of 2–600 ng/mL. The lower limit of quantification was 2 ng/mL. The method also exhibited satisfactory results in terms of sensitivity, specificity, accuracy (with relative error ranging from –4.0% to 1.1%), precision (with intra- and inter-day relative standard deviations ranging from 2.8% to 9.5%),matrix effect, recovery as well as stability. Taken together, our newly developed method was reliable to monitor sunitinib concentrations in rabbit plasma.     

Simultaneous determination of ivabradine and N-desmethylivabradine in human plasma and urine using a LC-MS/MS method: application to a pharmacokinetic study

A sensitive and specific liquid-chromatography tandem mass spectrometry (LC-MS/MS) assay has been developed and validated for the simultaneous quantification of ivabradine and its active metabolite N-desmethylivabradine in human plasma and urine. The assay employed a single liquid–liquid extraction of the analytes from plasma and urine samples, and diazepam was used as internal standard (IS). The chromatographic separation was achieved on a Diamonsil C₁₈ column (150 mm×4.6 mm, 5 μm, Dikma) using a mixture of methanol and aqueous 5 mM ammonium acetate buffer containing 0.2% formic acid (80:20, v/v) as mobile phase. The assay for ivabradine and N-desmethylivabradine in plasma showed good linearity (r≥0.99) over the ranges 0.1013–101.3 ng/mL and 0.085–25.5 ng/mL, respectively. The assay for ivabradine and N-desmethylivabradine in urine showed good linearity (r≥0.99) over the ranges 10.13–6078 ng/mL and 8.5–850 ng/mL, respectively. The intra- and inter-day accuracy and precision values were found to be within the assay variability limits (RSD<15%) in accordance with FDA guidelines. The methods were successfully used for evaluating the pharmacokinetic properties of ivabradine and N-desmethylivabradine in human plasma and urine in Chinese healthy volunteers.     

A simple and sensitive gradient elution liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the quantification of doxorubicin in rabbit plasma

In the present study, we developed and validated a simple and sensitive gradient elution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of doxorubicin in rabbit plasma. Daunorubicin was used as an internal standard (IS). The doxorubicin and IS were extracted with ethyl acetate from plasma samples. The chromatographic separations were achieved on a C₁₈ column (2.1 mm×50 mm, 2.5 μm) configured with a C₁₈ guard column (2.1 mm×10 mm, 2.5 μm). The mobile phase of 0.1% formic acid-water solution and acetonitrile was delivered using a gradient elution program at a flow rate of 0.4 mL/min. The temperature for column was maintained at 40 ºC. The electrospray ionization (ESI) source was operated in the positive ion mode, and the quantification was conducted using multiple reaction monitoring (MRM) of the transitions m/z 544.07→396.96 and m/z 528.06→321.05 for doxorubicin and IS, respectively. The calibration curve of doxorubicin was linear (r > 0.999) within the range of 2-600 ng/mL. The lower limit of quantification was 2 ng/mL. The relative errors of intra­day and inter-day accuracies ranged from -2.48% to 0.18% and from -3.78% to 1.94%, respectively. The relative standard deviations of intra­day and inter-day precisions were less than 8.65% and 6.41%, respectively. The method exhibited satisfactory results in terms of specificity, sensitivity, matrix effect, recovery and stability. The newly developed LC-MS/MS method was reliable to monitor doxorubicin concentrations in rabbit plasma.     

Determination of platinum complexes in clinical samples by a rapid flameless atomic absorption spectrometry assay

The frequent use of platinum (Pt) complexes in cancer chemotherapy and the application of new therapeutic options and dosing strategies have increased the need for rapid analytic procedures to determine Pt concentrations in the biologic fluids of patients. Therefore a flameless atomic absorption spectrometry method for the quantification of Pt in plasma and ultrafiltrate was developed and validated. A simple sample preparation of only one dilution step was established. Only 400 microL of whole blood was required for duplicate analysis of Pt in both matrices. The matrix-specific temperature programs took less than 75 seconds. The lower limit of quantification was 40 ng Pt/mL and 20 ng Pt/mL for plasma and ultrafiltrate, respectively. Suitable linearity could be reached using separate calibration curves for the high and low Pt concentration ranges. Recovery of Pt was complete, and there were no major stability problems. The accuracy and precision of the new method met the international criteria for the validation of bioanalytic methods. In addition, the use of different anticoagulants for clinical sampling, ultrafiltration systems, and ultrafiltration conditions were investigated. The assay has already been extensively applied to pharmacokinetic studies. In conclusion, the new Pt assay proved to be rapid, simple, sensitive, and suitable for clinical use.     

Simultaneous determination of GFA and its active metabolites in human plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies

A sensitive and rapid liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method has been developed and validated for simultaneous quantification of guanfu base A (GFA) and its metabolites guanfu base I (GFI) and guanfu alcohol-amine (AA) in human plasma with phenoprolamine hydrochloride (DDPH) as the internal standard. The analytes were extracted from human plasma by using liquid-liquid extraction with ethyl acetate and the LC separation was performed on a Diamonsil C(18) analytical column (150 mm x 2.1 mm i.d., 5 microm). The MS acquisition was performed in selected ion monitoring (SIM) mode of positive ions. Analysis was carried out in SIM mode at m/z 430.25 for GFA [M+H](+), m/z 388.25 for GFI [M+H](+), m/z 346.25 for AA [M+H](+) and m/z 344.20 for the IS DDPH [M+H](+). The calibration curves were linear over the range of 50-5000 ng/mL for GFA and 5-1000 ng/mL for GFI and AA, with coefficients of correlation above 0.999. The lower limit of quantification for GFA was 1 ng/mL, while for GFI and AA were both 5 ng/mL. The intra- and inter-day precisions (CV) of analysis were within 9%, and the accuracy ranged from 91% to 108%. The overall recoveries for GFA, GFI and AA were about 94.2%, 87.8% and 80.6%, respectively. The total LC-MS run-time was only 5.5 min. This quantitation method was successfully applied to the simultaneous determination of GFA and its metabolites in human plasma for the metabolic study and pharmacokinetic evaluation.     

Identification and quantification of trihexyphenidyl and its hydroxylated metabolite by gas chromatography with nitrogen-phosphorus detection

A sensitive and specific assay for the simultaneous quantification of trihexyphenidyl and its hydroxylated metabolite in plasma and urine is described. The method is based on the extraction of the drugs with an organic solvent and separation on a 3% OV-17 Chromosorb Q column in a gas chromatograph equipped with a nitrogen-phosphorus detector. The procedure employs SKF 525 A as the internal standard and requires no derivatization. The detection limit was found to be 2 ng/mL for trihexyphenidyl and 1 ng/mL for its metabolite. The precision of the assay procedure for both compounds is about 4 to 7%.     

Development of chiral liquid chromatography-tandem mass spectrometry isotope dilution methods for the determination of unconjugated and total S-equol in human plasma and urine

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of unconjugated and total (conjugated plus unconjugated) S-equol in human plasma and urine were developed and validated. The separation of R and S enantiomers was achieved with a Chiracel OJ-H column operated in a normal phase mode using ethanol/hexane mobile phase components. Ionization of S-equol by negative ion electrospray generated the [M-H](-) ion whose response was augmented by post-column addition of ammonium hydroxide. A triple stage quadrupole mass spectrometer was used to measure the ion current generated from the dissociative transitions m/z 241→m/z 121 (S-equol) and m/z 245→m/z 123 (equol-d(4)). The determination of total S-equol included an additional deconjugation step involving incubation of the sample with sulfatase and glucuronidase. Average recovery for both unconjugated and total S-equol was 85% with no observable matrix effects. Linearity was established for unconjugated S-equol from 0.025ng/mL to 10ng/mL (plasma) and 0.20ng/mL to 200ng/mL (urine). The average coefficient of variation and accuracy per occasion was within ±15% of the theoretical concentration of S-equol. The method was used to measure the pharmacokinetics of S-equol in human plasma after an oral administration of a single 20mg dose of S-equol to three normal healthy volunteers.     

Liquid chromatographic-electrospray tandem mass spectrometric method for the quantification of nimodipine in human plasma

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of nimodipine, a calcium channel blocker, in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 419/343 for nimodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 0.2-50 ng/mL for nimodipine in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid determination of granisetron in human plasma by liquid chromatography coupled to tandem mass spectrometry and its application to bioequivalence study

A simple, sensitive and rapid method for analysis of granisetron in human plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and validated to satisfy FDA guidelines for bioanalytical methods. The analyte and internal standard (IS) were isolated from 100microl plasma samples by liquid-liquid extraction (LLE). A Varian 1200l tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 313.4/138 for granisetron and m/z 270/201 for the IS used for quantitation. The assay exhibited a linear dynamic range of 0.02-20ng/ml for granisetron in human plasma. The lower limit of quantification (LLOQ) was 0.02ng/ml with a relative standard deviation of less than 15%. The mean extraction recovery from spiked plasma samples was 97.9%. The intra-day accuracy of the assay was within 10% of nominal and intra-day precision was better than 15% C.V. A run time of 2.0min for each sample made it possible for high-throughput bioanalysis. The method was employed in a bioequivalence study of two formulations of granisetron hydrochloride 1mg rapidly disintegrating tablets/1mg capsules.