Quantitative determination of azithromycin in human plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry and its application in a pharmacokinetic study

A selective, rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the quantitative determination of azithromycin in human plasma and its application in a pharmacokinetic study. With roxithromycin as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.5 mL plasma. The analysis was carried out on an ACQUITY UPLC BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with gradient elution at flow rate of 0.35 mL/min. The mobile phase was 50 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 1-1000 ng/mL, with a lower limit of quantification of 1 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was -1.3% to 5.7% at all QC levels. The method was applicable to clinical pharmacokinetic study of azithromycin in healthy volunteers following oral administration.     

Highly sensitive determination of estrone and estradiol in human serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A highly sensitive and specific quantification method of estrone and estradiol in human serum was described based upon the use of picolinoyl derivatization and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) in a positive mode. Estrogens were treated with picolinoyl chloride hydrochloride or picolinic acid and 2-methyl-6-nitrobenzoic anhydride followed by a solid-phase extraction with ODS cartridge. Picolinoyl derivatization proceeded quantitatively even in a microscale, and the picolinoyl esters provided simple positive ESI-mass spectra showing [M+H](+) as base peaks for these estrogens. The picolinoyl derivatives of these estrogens showed 100-fold higher detection response compared to underivatized intact molecules by LC-ESI-MS (selected reaction monitoring). Using this derivatization, estrogens spiked in the charcoal treated human serum samples were analyzed with limit of quantification (LOQ), intra-day accuracy and precision of 1.0pg/ml, 96.0% and 9.9% for estrone, and 0.5pg/ml, 84.4% and 12.8% for estradiol, respectively. Estrone and estradiol added to the crude serum samples were recovered with comparable LOQ and accuracy obtained for the charcoal treated serum samples as well.     

Determination of talinolol in human plasma by high performance liquid chromatography-electrospray ionization mass spectrometry: application to pharmacokinetic study

A rapid and sensitive method for determination and screening in human plasma of talinolol is described using propranolol as the internal standard. The analytes in plasma were extracted by liquid-liquid extraction using methyl t-butyl ether. After removed and dried the upper organic phase, the extracts were reconstituted with a fixed volume of buffer of ammonium acetate and acetonitrile (60:40, v/v). The extracts were analyzed by a HPLC coupled to electrospray ionization mass spectrometry (HPLC-MS/ESI). The HPLC separation of the analytes was performed on a Phenomenex C18 (250 mmx4.6 mm, 5 microm, USA) column, with a flow rate of 0.85 mL/min. The complete elution was obtained within 5.5 min. The calibration curve was linear in the 1.0-400.0 ng/mL range for talinolol, with a coefficient of determination of 0.9996. The average extraction recovery was above 83%. The methodology recovery was between 101% and 102%. The limit of detection (LOD) was 0.3 ng/mL for talinolol. The intraday and inter-day coefficients of variation were less than 6%. This HPLC-MS/ESI procedure was used to assess the pharmacokinetics of talinolol. A single oral 50 mg dose of talinolol tablet was administered to 12 healthy Chinese volunteers, the main pharmacokinetic data are as follows: Cmax was 147.8+/-63.8 ng/mL; tmax was 2.0+/-0.7 h; t1/2 was 12.0+/-2.6 h. The method is accurate, sensitive and simple for the pharmacokinetic study of talinolol.     

Determination of glimepiride in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS-MS) method has been developed at our center for the determination of glimepiride in human plasma. After the addition of the internal standard, plasma samples were extracted by liquid-liquid extraction technique using diethyl ether. The compounds were separated on a prepacked C18 column using a mixture of acetonitrile, methanol and ammonium acetate buffer as mobile phase. A Finnigan LCQDUO ion trap mass spectrometer connected to an Alliance Waters HPLC was used to develop and validate the method. The analytical method was validated according to the FDA bioanalytical method validation guidance. The results were within the accepted criteria as stated in the aforementioned guidance. The method was proved to be sensitive and specific by testing six different plasma batches. Linearity was established for the range of concentrations 5.0-500.0 ng/ml with a coefficient of determination (r2) of 0.9998. Accuracy for glimepiride ranged from 100.58 to 104.48% at low, mid and high levels. The intra-day precision was better than 12.24%. The lower limit of quantitation (LLOQ) was identifiable and reproducible at 5.0 ng/ml with a precision of 7.96%. The proposed method enables the unambiguous identification and quantitation of glimepiride for pharmacokinetic, bioavailability or bioequivalence studies.     

Determination of bulleyaconitine A in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid-liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor-->product ion combinations at m/z 644.6-->584.3 and 531.2-->81.6, respectively. Linearity was established for the concentration range of 0.12-6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.     

Quantitative determination of helicid in rat plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to preliminary pharmacokinetic studies

A sensitive and selective liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the identification and quantification of helicid in rat plasma. The method was based on simple liquid-liquid extraction (LLE). A Kromasil C18 column (150mmx2.00mm, 3.5microm) was used as the analytical column, while a mixture of acetonitrile and 500microM ammonium chloride was used as the mobile phase. MS detection was performed using a single quadrupole mass spectrometer in a negative selected ion monitoring (SIM) mode. The deprotonated molecules [M+Cl](-) at m/z 319.00 and 363.05 were used to quantify helicid and bergeninum (internal standard, I.S.), respectively. The lower limit of quantification of helicid was 1ng/ml. The method was linear in the concentration range of 1-1000ng/ml. The intra-day and inter-day precisions (R.S.D.%) were within 10.0% for the analyte. Helicid proved to be stable during all sample storage, preparation and analytical periods. The method was successfully applied to a pharmacokinetic study in rats after intragastric administration of helicid with a dose of 50mg/kg. Only 50microl of rat plasma at each sampling time was needed for analysis. The proposed method enables unambiguous identification and quantification for the preliminary pharmacokinetic studies of helicid.     

Determination of levocetirizine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study

We describe a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for levocetirizine quantification (I) in human plasma. Sample preparation was made using a fexofenadine (II) addition as internal standard (IS), liquid-liquid extraction using cold dichloromethane, and dissolving the final extract in acetonitrile. I and II (IS) were injected in a C18 column and the mobile phase composed of acetonitrile:water:formic acid (80.00:19.90:0.10, v/v/v) and monitored using positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 389>201 for I and m/z 502>467 for II. The limit of quantification and the dynamic range achieved were 0.5ng/mL and 0.5-500.0ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples taken up to 48h after oral administration of 5mg of levocetirizine dichloridrate in healthy volunteers demonstrate its applicability to bioavailability studies.     

Determination of memantine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of memantine (I) in human plasma is presented. Sample preparation consisted of the addition of amantadine (II) as internal standard (IS), liquid-liquid extraction in basic conditions using a mixture of diethyl ether-chloroform (7:3, v/v) as extracting solvent, followed by centrifugation, solvent evaporation and sample reconstitution in methanol. Both I and II (internal standard) were analyzed using a C18 column and a mobile phase composed of methanol-water-formic acid (80:20:0.1, v/v/v). Eluted compounds were monitored using positive mode electrospray (ES) tandem mass spectrometry. The analyses were carried out by selected reaction monitoring (SRM) using the parent to daughter combinations of m/z 180>163 (memantine) and m/z 152>135 (amantadine). The peak areas from the analyte and IS were used for quantification of I. The achieved limit of quantification (LOQ) was 0.1 ng/mL; the assay exhibited a linear dynamic range of 0.1-50.0 ng/mL with a determination coefficient (r2) of at least 0.98. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of samples taken up to 320 h after oral administration of 20mg (two 10mg capsules) of I in healthy volunteers demonstrated the applicability to bioequivalence studies.     

Development and validation of a sensitive liquid chromatography/tandem mass spectrometry method for the determination of exemestane in human plasma

Exemestane, irreversible steroidal aromatase inhibitor, acts as a false substrate for aromatase enzyme and significantly lowers circulating estrogen concentrations in postmenopausal women with hormone-sensitive breast cancer. A sensitive bioanalytical method was developed and validated to study pharmacokinetics of exemestane. The method was based on liquid-liquid extraction of exemestane with methyl t-butyl ether followed by reversed-phase liquid chromatography. Positive electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode was applied for detection of exemestane. Anastrozole was used as internal standard. Calibration curve, fitted to 1/x2 weighted linear regression model, was linear in the range of 0.1-40.0 ng/mL. Intra-run precision and accuracy were 1.80-3.17% and 103.4-111.5%, respectively. Inter-run precision and accuracy measured within 3 days were 3.37-4.19% and 101.8-109.6%, respectively. Extraction recoveries of exemestane and internal standard were 79.7-86.2% and 82.9-83.6%, respectively. The method was fully validated and may be applied to pharmacokinetic studies in humans after a single dose administration of 25mg exemestane tablets.     

Simultaneous determination of metformin and rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: application to a pharmacokinetic study

A selective and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous determination of metformin and rosiglitazone in human plasma using phenformin as internal standard (IS) has been first developed and validated. Plasma samples were precipitated by acetonitrile and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A (150 mm x 2.0 mm I.D.) column using a mobile phase comprised of methanol:30 mM ammonium acetate pH 5.0 (80:20, v/v) delivered at 0.2 ml/min. Detection was performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in positive ion selected reaction monitoring (SRM) mode using electrospray ionization. The ion transitions monitored were m/z 130.27-->71.11 for metformin, m/z 358.14-->135.07 for rosiglitazone and m/z 206.20-->105.19 for the IS. The standard curves were linear (r(2)>0.99) over the concentration range of 5-3000 ng/ml for metformin and 1.5-500 ng/ml for rosiglitazone with acceptable accuracy and precision, respectively. The within- and between-batch precisions were less than 15% of the relative standard deviation. The limit of detection (LOD) of both metformin and rosiglitazone was 1 ng/ml. The method described is precise and sensitive and has been successfully applied to the study of pharmacokinetics of compound metformin and rosiglitazone capsules in 12 healthy Chinese volunteers.     

Determination of dimenhydrinate in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a relative bioavailability study

Here we present a sensitive and specific liquid chromatography-tandem mass spectrometric method for the quantification of dimenhydrinate (I) in human plasma. Sample preparation is conducted using citalopram (II) addition as an internal standard (IS), liquid-liquid extraction with basified plasma using a mixture hexane/acetate (1:1, v/v) as the extracting solvent, and the final extract reconstituted in the mobile phase. I and II (IS) were injected in a C8 column with the mobile phase composed of methanol:isopropanol:water:formic acid (78.00:19.92:2.00:0.08, v/v/v/v) and monitored using a positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 256.0>167.0 and m/z 325.0>109.0 for I and II, respectively. The limit of quantification (LOQ) was 0.4 ng/mL, the dynamic range being 0.4-200 ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as on application to the analysis of plasma samples taken up to 24 h after oral administration of 100 mg of dimenhydrinate in healthy volunteers demonstrated its applicability to bioavailability studies.     

Lisinopril quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00-200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n = 8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20mg.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of phenylephrine in human plasma and its application to a pharmacokinetic study

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography-electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm x 4 mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1-->135.0 for phenylephrine and m/z 182.1-->135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Quantitation of tryptophan,kynurenine and kynurenic acid in human plasma by capillary liquid chromatography-electrospray ionization tandem mass spectrometry

Concentrations of tryptophan and its metabolites in plasma are of great interest in determining proper diagnosis and medication of several neurological diseases like, for example, Alzheimer's disease. A method of standard addition was developed to determine total level of tryptophan and two of its metabolites, kynurenine and kynurenic acid, in human plasma by capillary liquid chromatography-electrospray ionization tandem mass spectrometry. Plasma samples were simply deproteinized by addition of diluted perchloric acid. Samples were then mixed with trichloroacetic acid and injected onto a capillary column. Analytes were separated by a fast gradient elution of the injected samples. Detection was performed by sheathless electrospray tandem mass spectrometry in the multiple reaction monitoring mode. Linear calibration curves were obtained for spiked plasma sample with up to 100% of the expected analytes concentrations. The determined concentrations were well within ranges previously reported (i.e., 6 nM-95 microM) and limit of detections were around 3 nM for each analyte.     

Determination of misoprostol acid in human plasma by liquid chromatography coupled to tandem mass spectrometry

A rapid and sensitive liquid chromatographic/tandem mass spectrometric method for determination of misoprostol acid, the active metabolite of misoprostol, was developed and validated. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a C(18) column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 367-249 and 296-269 was performed to quantify misoprostol acid and the internal standard hydrochlorothiazide, respectively. The method was linear in the concentration range of 10.0-3000 pg mL(-1) using 200 microL plasma. The lower limit of quantification was 10.0 pg mL(-1). The intra- and inter-day relative standard deviation over the entire concentration range was less than 8.3%. Accuracy determined at three concentrations (25.0, 200 and 2700 pg mL(-1) for misoprostol acid) ranged from -0.5 to 1.2% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method was successfully used in a pharmacokinetic study of misoprostol in human plasma after an oral administration of 0.6 mg misoprostol.     

Lansoprazole quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of lansoprazole in human plasma using omeprazole as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction using diethyl-ether-dichloromethane (70:30; v/v) and chromatographed on a C(18) analytical column. The mobile phase consisted of acetonitrile-water (90:10; v/v)+10 mM formic acid. The method has a chromatographic total run time of 5 min and was linear within the range 2.5-2000 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by Multiple Reaction Monitoring (MRM). The intra- and inter-run precision, calculated from quality control (QC) samples, was less than 3.4%. The accuracy as determined from QC samples was less than 9%. The method herein described was employed in a bioequivalence study of two capsule formulations of lansoprazole.     

Quantitative determination of fluorinated caffeic acid phenethyl ester derivative from rat blood plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

The quantitative determination of caffeic acid phenethyl ester (CAPE) and its fluorinated derivative (FCAPE) from rat plasma using ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is reported. CAPE and FCAPE were extracted using ethyl acetate in the presence of methyl caffeate (MC) as internal standard. Separation was achieved using a C(18) column (2.1 mm x 50 mm, 1.7 microm) and gradient elution with water and acetonitrile containing 0.2% and 0.1% formic acid, respectively. A non-linear response over a broad concentration range (1-1000 ng/ml, r(2)>0.995 using a quadratic regression model and 1/concentration weighting) was obtained. The inter-day and intra-day variability for CAPE and FCAPE were found to be less than 14.2% and 9.5%, respectively. Data are presented to illustrate the practicality of the method for the pharmacokinetic evaluation of CAPE and FCAPE after intravenous administration to rats.     

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.     

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.     

Determination of ziprasidone in human plasma by liquid chromatography-electrospray tandem mass spectrometry and its application to plasma level determination in schizophrenia patients

An accurate, rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay method was developed for the determination of ziprasidone (ZIP) in the plasma of schizophrenia patients. A simple one step liquid-liquid extraction with 20% methylene dichloride in pentane was used to isolate ZIP and the internal standard from the plasma matrix. The compounds were separated on a C-18 column by an isocratic elution and the eluted compounds were analyzed by a triple quadrupole mass spectrometer with a TurboIon spray interface using the positive ion atmospheric pressure electrospray ionization method and detected using multiple reaction monitoring mode. The ZIP standard calibration curve was linear over the range of 0.25-500ng/ml when 0.5ml of plasma was used for the analysis (r(2)>0.998). The intra-assay (within-day) and inter-assay (between-day) variations were less than 12% for the spiked standard curve and quality control samples. The absolute extraction efficiency was 82% for ZIP and 68% for INS-RSP. The analysis time for each sample was less than 3min and useful for high turnaround plasma level determinations. This LC-MS-MS assay method for ZIP is highly specific, sensitive, accurate and rapid and is currently being used for the plasma level determination of ZIP in schizophrenia patients treated with various daily oral doses of ZIP. The data showed large inter-individual variations.