Rapid quantification of lamotrigine in human plasma by two LC systems connected with tandem MS.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the determination of lamotrigine in human plasma using multiplexing technique (two HPLC units connected to one MS-MS). Lamotrigine was extracted from human plasma by solid-phase extraction technique using Oasis Hydrophilic Lipophilic Balance (HLB) or N-vinylpyrrolidone and divinylbenzene cartridge. A structural analog, 3,5-diamino-6-phenyl-1,2,4-triazine, was used as an internal standard (IS). A BetaBasic C(8) column was used for the chromatographic separation of analytes. The mass transition [M+H](+) ions used for detection were m/z 256.0 --> 211.0 for lamotrigine and m/z 188.0 --> 143.0 for IS. The method involved a simple multiplexing, rapid solid-phase extraction without evaporation and reconstitution. The proposed method has been validated for a linear range of 0.025 to 10.000 microg/mL with a correlation coefficient > or = 0.9991. The limit of quantification for lamotrigine was 0.025 microg/mL, and limit of detection was 50.000 pg/mL. The intra-run and inter-run precision and accuracy were within 10.0% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for lamotrigine and IS were 97.9% and 92.5%, respectively. Total MS run time was 1.4 min per sample. The validated method has been successfully used to analyze human plasma samples for applications in pharmacokinetic, bioavailability, bioequivalence, or in vitro in vivo correlation studies.

Quantification of paroxetine in human plasma by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A rapid LC coupled with electrospray ionization (ESI) MS/MS method was developed and validated for the quantification of paroxetine in heparinized human plasma. The plasma samples were prepared by the solid-phase extraction method without drying or reconstitution. Elution was done with 0.5 mL 0.2% (v/v) formic acid in methanol-acetonitrile (65 + 35, v/v). The analyte and the internal standard (IS; imipramine hydrochloride) were chromatographed on a BDS Hypersil C18 column. The analyte was analyzed by LC/MS/MS with only 1.7 min run time. An ESI interface was chosen for ionization of the analyte from the sample matrix. Selected reaction monitoring mode for detection of paroxetine and the IS were achieved by using m/z 330.17/192.10 and 281.13/86.14, respectively. The LC retention times for paroxetine and imipramine were 0.94 and 1.05 min, respectively. The method was linear in the concentration range of 0.5-80.0 ng/mL with r > or = 0.9995. Recovery of paroxetine and imipramine ranged from 90 to 95%. The assay has been successfully applied to bioequivalence study samples for estimation of paroxetine in healthy human subjects.

In vitro-in vivo correlation of modified release dosage form of lamotrigine.

The plasma concentration profile of lamotrigine was predicted from the dissolution test data of the modified release 100 mg lamotrigine tablet by applying the in vitro-in vivo correlation (IVIVC). Three different release formulations (L-1, L-2 and L-3) and its profiles of in vitro data were generated in different dissolution media. Pharmacokinetics evaluation of these formulations was carried out in 12 healthy volunteers. In vitro-in vivo correlation was established from the generated dissolution and bioavailability data. A good correlation between the percentages dissolved vs absorbed (r2>0.989) was obtained using level A correlation. Evaluation of the internal predictability of level A correlation was calculated in terms of percent prediction error, which was found to be below 15%.

Rapid determination of losartan and losartan acid in human plasma by multiplexed LC-MS/MS.

A rapid LC-MS/MS method has been developed and validated for the determination of losartan (LOS) and its metabolite losartan acid (LA) (EXP-3174) in human plasma using multiplexing technique (two HPLC units connected to one MS/MS). LOS and LA were extracted from human plasma by SPE technique using Oasis HLB cartridge without evaporation and reconstitution steps. Hydroflumethiazide (HFTZ) was used as an internal standard (IS). The analytes were separated on Zorbax SB C-18 column. The mass transition [M-H] ions used for detection were m/z 421.0 --> 127.0 for LOS, m/z 435.0 --> 157.0 for LA, and m/z 330.0 --> 239.0 for HFTZ. The proposed method was validated over the concentration range of 2.5-2000 ng/mL for LOS and 5.0-3000 ng/mL for LA with correlation coefficient > or = 0.9993. The overall recoveries for LOS, LA, and IS were 96.53, 99.86, and 94.16%, respectively. Total MS run time was 2.0 min/sample. The validated method has been successfully used to analyze human plasma samples for applications in 100 mg fasted and fed pharmacokinetic studies.

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.

Rapid LC-ESI-MS-MS method for the simultaneous determination of clopidogrel and its carboxylic acid metabolite in human plasma.

A high throughput liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) method is developed for the simultaneous estimation of clopidogrel (SR25990C) and its carboxylic acid metabolite (SR26334) in human plasma using glimepiride as internal standard. The extraction of SR25990C, its metabolite, and IS from the plasma (0.3 mL) involves treatment with phosphoric acid followed by solid-phase extraction (SPE). Sample preparation by this method yields clean extracts with quantitative and consistent mean recoveries of 98.05%, 85.45%, and 105.72% for SR25990C, SR26334, and IS, respectively. The SPE eluate without drying and reconstitution is analyzed by LC-MS-MS, operating in the positive ion and selective reaction monitoring mode. The injection volume is 2 microL with a total chromatographic run time of 5.0 min. The method response is linear over the dynamic range of 0.25 to 25.0 ng/mL for SR25990C and 50.0 to 6000.0 ng/mL for SR26334, with correlation coefficients of r > or = 0.9989 and 0.9984, respectively. The method is validated to demonstrate its specificity, linearity, accuracy, precision, recovery, matrix effect, dilution integrity, and stability studies. It is applied to study the bioavailability of 75 mg clopidogrel mesylate tablets in 16 human subjects with satisfactory results.

Direct injection, column switching-liquid chromatographic technique for the estimation of rabeprazole in bioequivalence study.

A rapid, simple and sensitive high-performance liquid chromatography-ultra violet (HPLC-UV) method with column switching between sample pre-treatment column and analytical column was developed for the quantitation of rabeprazole in human plasma; on a Bio-Sample Analysis system (Co-sense for BA) from Shimadzu Corporation, Kyoto, Japan. Zaleplon was used as an internal standard. The method was validated as per USFDA guidelines for the concentration range of 20.0-1200.0 ng/mL and the correlation coefficient were found to be better than 0.999. Recovery of rabeprazole as well as the internal standard from human plasma was more than 90.0%. Rabeprazole was stable in human plasma for 4 months at -70 +/- 5 degrees C and for 20.0 h at ambient temperature. In the auto sampler, the drug was stable for 24.0 h at 4 degrees C. The method was specific as there were no interfering peaks in the human plasma eluting at the retention times of the rabeprazole and the internal standard. The frozen plasma samples containing rabeprazole were stable to three freeze thaw cycles. The bioanalytical method was rugged in terms of inter- and intra-day accuracy and precision. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of 20mg rabeprazole tablet of German Remedies Ltd. (A division of Cadila Healthcare Ltd.), India versus Pariet tablet of Eisai Ltd. & Janssen-Cilag Ltd., Japan in male human subjects.

Liquid chromatography--electrospray ionisation mass spectrometry method for the determination of escitalopram in human plasma and its application in bioequivalence study.

A novel liquid chromatographic--electrospray ionisation mass spectrometric (LC--ESI-MS) method has been developed for the determination of escitalopram, an antidepressant in human plasma using paroxetine as internal standard. The method involved liquid--liquid extraction of the analyte from human plasma with a mixture of diethyl ether and dichloromethane (70:30, v/v). The chromatographic separation was achieved within 7.0 min by using 2.0 mM ammonium acetate (pH 5.0)--acetonitrile (54:46, v/v) as mobile phase and a ODS YMCAQ 150 mm x 4.6 mm analytical column; the flow-rate was 1.0 ml/min. Ion signals m/z 325.0 and 330.0 for escitalopram and internal standard, were measured in the positive mode. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range of 1.0-200 ng/ml with a mean correlation coefficient more than 0.99. The absolute recovery was more than 75% for both escitalopram and internal standard. The method was simple, sensitive, precise, accurate and was successfully applied to the bioequivalence study of escitalopram in healthy, male, human subjects.

Validation of LC/MS electrospray ionisation method for the estimation of ursodiol in human plasma and its application in bioequivalence study.

A novel High Performance Liquid Chromatography-electrospray mass spectrometric method has been developed for the estimation of Ursodiol (Ursodeoxycholic acid)--a bile acid, in human plasma using Ornidazole as internal standard. The methodology involved solid phase extraction of the analyte from human plasma matrix. The chromatographic separation was achieved within seven minutes by an isocratic mobile phase containing 1.0 mM ammonium acetate and Acetonitrile (65:35, v/v), flowing through XTerra MS C18, 100 x 2.1, 3.5 microm analytical column, at a flow rate of 0.2 ml/min. Ion signals were measured in negative mode for Ursodiol and internal standard at m/z 391.3 and 278.1, respectively. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range 50.0 ng/ml to 3000.0 ng/ml with the mean correlation coefficient more than 0.99. The absolute recovery was more than 54.90% for Ursodiol and 76.51% for internal standard. Ursodiol was stable for sixty-nine days at -70 degrees C and for eight hours at ambient temperature. After extraction from plasma, the reconstituted samples of Ursodiol were stable in autosampler at 10 degrees C for forty-eight hours. Upon subjecting to three freeze thaw cycles, there was no change in the recovery of the analyte. The integrity of the plasma samples remained unaffected even upon four-fold dilution with drug free human plasma. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of Ursodiol in male human subjects.