Simultaneous determination of rivanol and mifepristone in human plasma by a HPLC-UV method with solid-phase extraction

A HPLC method with UV detection was developed and validated for the simultaneous determination of rivanol and mifepristone in human plasma. Norethisterone was used as the internal standard. Separation was performed by a C18 reversed-phase column maintained at 20 degrees C. The mobile phase was a mixture of methanol-acetonitrile-0.05% sodium dodecylsulfonate in a 0.05 M phosphate buffer with the pH adjusted to 3.0 (30:30:40, v/v/v) at a flow rate of 0.8 ml/min. Dual wavelength mode was used, with mifepristone monitored at UV 302 nm, while rivanol and norethisterone at 272 nm. A reliable biological sample pre-treatment procedure by means of solid-phase extraction was used, which allowed to obtain good extraction efficiency (>93%) for both of the analytes and the internal standard. The calibration curves were both linear with the correlation coefficient r equal to 0.9999. For rivanol, the assay gave CV% values for precision always lower than 7.8% and mean accuracy values higher than 95.3%. As to mifepristone, precision was always lower than 10.1% and mean accuracy values were higher than 93.8%. The limit of detection for the assay of rivanol and mifepristone was 1.1 and 3 ng/ml, respectively. The method is simple, sensitive and accurate, and allow for simultaneous determination of nanogram levels of rivanol and mifepristone in human plasma. It could be applied to assess the plasma level of rivanol and mifepristone in women undergoing polypharmacy with the two drugs.     

Simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) procedure for the simultaneous determination of quinapril and its active metabolite quinaprilat in human plasma samples is described. A one-step solid-phase extraction (SPE) with C18 cartridges was coupled with a reversed-phase HPLC system. The system requires two mobile phases composed of tetrabutyl ammonium hydrogensulfate (10 mM adjusted to pH 7)-acetonitrile (62:38, v/v) for quinapril, and (25:75, v/v) for quinaprilat elution through a C18 Symmetry column and detection at a wavelength of 215 nm. Calibration curves were linear over the ranges 20 to 1,000 ng/ml for quinaprilat and 10 to 500 for quinapril. The limits of quantification were 20 and 10 ng/ml for quinaprilat and quinapril, respectively. Extraction recoveries were higher than 90% for quinapril and 80% for quinaprilat. This method has been successfully applied to a bioequivalence study of quinapril in healthy subjects.     

Rapid determination of loratadine in small volume plasma samples by high-performance liquid chromatography with fluorescence detection

A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid-liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62-20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Simultaneous determination of nimesulide and hydroxynimesulide in rat plasma,cerebrospinal fluid and brain by liquid chromatography using solid-phase extraction

A liquid chromatographic method with UV detection for the quantification of nimesulide (N) and hydroxynimesulide (M1) in rat plasma, cerebrospinal fluid (CSF) and brain tissue is reported. Plasma samples (250 microl) and brain homogenates added with the right amount of the internal standard (I.S., 2'-(cyclohexyloxy)-4'-nitrophenyl methanesulphonanilide, NS398) are extracted on C(18) disposable cartridges by solid-phase extraction (SPE), while CSF samples are analyzed without any extraction. The separation is performed at room temperature on a Waters Symmetry C(18) 3.5 microm (150x4.6 mm I.D.) column with acetonitrile-sodium citrate buffer pH 3.00 (53:47, v/v) as mobile phase, at a flow-rate of 1.1 ml/min and detection at 240 nm. The retention times are 3.3, 6.0 and 9.9 min for M1, N and I.S., respectively. The lower limits of quantitation for either nimesulide and M1 are 25 ng/ml for plasma, 20 ng/ml for CSF and 25 ng/g for brain tissue. The calibration curves are linear up to 10,000 ng/ml for plasma, 5000 ng/ml for CSF and 5000 ng/g for brain tissue. This new assay can be applied to the study of the role of nimesulide in the modulation of neuroinflammatory processes.     

Rapid determination of ranitidine in human plasma by high-performance liquid chromatography without solvent extraction

A simple high-performance liquid chromatographic procedure was developed for the determination of ranitidine in human plasma. The method entailed direct injection of the plasma samples after deproteination using perchloric acid. The chromatographic separation was accomplished with an isocratic elution using mobile phase consisting of 21 mM disodium hydrogen phosphate–triethylamine-acetonitrile (1000:60:150, v/v), pH 3.5. Analyses were run at a flow-rate of 1.3 ml/min using a μbondapak C₁₈ column and ultraviolet detection at a wavelength of 320 nm. The method was specific and sensitive, with a quantification limit of approximately 20 ng/ml and a detection limit of 5 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery was about 96%, while the within- and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The linearity was assessed in the range of 20–1000 ng/ml plasma, with a correlation coefficient of greater than 0.999. This method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 μm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250×4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3–100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%–94.4% and the mean inter-assay precision was 2.8%–3.2% (range 0.3–100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at −20°C for 4.3 months and at −80°C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.     

Determination of free serum didanosine by ultrafiltration and high-performance liquid chromatography

An isocratic reversed-phase high-performance liquid chromatographic method was developed to determine free didanosine concentrations in human serum. An ultrafiltration technique was used to recover didanosine from the samples. Didanosine was analyzed using a 150 mm × 3.9 mm I.D. Nova-Pak phenyl column and a mobile phase of 0.02 M sodium citrate (pH 5)-isopropanol (97.5:2.5, v/v) with detection set at 250 nm. Linearity was verified from 25 to 3000 ng/ml. The limit of detection at a signal-to-noise ratio of 3 was 25 ng/ml. The mean recovery of didanosine added to serum at 50, 100, 250 and 750 ng/ml was 97.4%, 97.3%, 92.9% and 95.4%, respectively. A within-day variation of 3.6% at 50 ng/ml and 1.7% at 250 ng/ml, and a day-to-day variation of 9.3% at 50 ng/ml and 3.6% at 230 ng/ml were found. Stability studies indicated that didanosine is stable in serum for at least 8.5 months at 20°C, 4°C and −20°C.     

High-performance liquid chromatographic analysis of DA-7867, a new oxazolidinone,in human plasma and urine and in rat tissue homogenates

An HPLC method was developed for the determination of a new oxazolidinone, DA-7867 (I), in human plasma and urine and in rat tissue homogenates. To 100 microl of biological sample, 300 microl acetonitrile and 50 microl methanol containing 10 microg/ml DA-7858 (the internal standard) were added. After vortex-mixing and centrifugation, the supernatant was evaporated under a gentle stream of nitrogen. The residue was reconstituted in 100 microl of the mobile phase and a 50-microl aliquot was injected directly onto the reversed-phase (C(18)) column. The mobile phase, 20 mM KH2PO4:acetonitrile (75:25, v/v) was run at a flow rate of 1.5 ml/min and the column effluent was monitored by a UV detector set at 300 nm. The retention times of I and DA-7858 were approximately 6.5 and 8.7 min, respectively. The detection limits of I in human plasma and urine and in rat tissue homogenates were 20, 20, and 50 ng/ml, respectively.     

Determination of mifepristone levels in wild canid serum using liquid chromatography

An HPLC method was developed to determine levels of mifepristone, in coyote (Canis latrans) serum where mifepristone will be used as an oral contragestive agent for nonlethal predator control. Serum samples were extracted using C(18) solid-phase extraction cartridges. A synthetic analog of mifepristone, RTI-3021-003, was used as the internal standard. Separation of the compounds was achieved by using a C(18) (150 x 4.6 mm) column. The mobile phase was 55% acetonitrile in water running at 1.0 ml/min with UV detection at 305 nm. The assay was linear in the range of 10 to 1000 ng/ml. Inter-day accuracies for 10, 200 and 1000 ng/ml were 95.9, 99.4 and 104.7%, respectively. Inter-day precisions measured by RSD were 19.8, 9.7 and 4.5%. Intra-day accuracies were 117, 106.9 and 99.4% for 10, 200 and 1000 ng/ml, respectively. Intra-day RSDs were 19.7, 3.7 and 9.3%, respectively. A simple, sensitive and validated HPLC analytical method was developed to quantitate mifepristone in canine serum.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Sensitive determination of clarithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A rapid, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of clarithromycin in human plasma. Liquid-liquid extraction of clarithromycin and norverapamil (as internal standard) from plasma samples was performed with n-hexane/1-butanol (98:2, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a CN column (250 mm x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-50 mM aqueous sodium dihydrogen phosphate (32:68, v/v), pH 4.5. Detection was made at 205 nm and analyses were run at a flow-rate of 1.0 ml/min at 40 degrees C. The analysis time was less than 11 min. The method was specific and sensitive with a quantification limit of 31.25 ng/ml and a detection limit of 10 ng/ml in plasma. The mean absolute recovery of clarithromycin from plasma was 95.9%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 9.5%. Linearity was assessed in the range of 31.25-2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method was used to analyze several hundred human plasma samples for bioavailability studies.     

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

Determination of the new podophyllotoxin derivative NK 611 in plasma by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method has been developed for the determination of the new podophyllotoxin derivative NK 611 in plasma samples. A solid—liquid extraction procedure with C₁₈ extraction columns was used for extraction of plasma samples containing NK 611. The adsorbed NK 611 was eluted from the extraction columns with methanol—acetonitrile (50:50, v/v). The elution liquid was injected into a reversed-phase system consisting of a Chrompack C₁₈ column. The mobile phase was acetonitrile—20 mM phosphate buffer, pH 7 (30:70, v/v). The UV detection mode allows sensitive determination of NK 611 in plasma within phase I trials. The limit of detection was 10 ng/ml, the limit of quantitation 35 ng/ml (for 1 ml of extracted plasma and 20-μl injection volume). The calibration curve is linear within the concentration range 100–1000 ng/ml. The recovery of NK 611 from spiked plasma samples was approximately 80%.     

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.     

Determination of ticlopidine in human plasma by high-performance liquid chromatography and ultraviolet absorbance detection

A simple HPLC method has been developed for the determination of ticlopidine in human plasma. Plasma samples were buffered at pH 9 and extracted with n-heptane-isoamyl alcohol (98.5: 1.5, v/v). Imipramine was used as internal standard. Chromatography was performed isocratically with acetonitrile-methanol-0.05 M KH₂PO₄ (20:25:55, v/v) at pH 3.0 containing 3% triethylamine at a flow-rate of 1 ml/min. A reversed-phase column, Supelcosil LC-8-DB, 15 cm × 4.6 mm I.D., 5 μm particle size, was used. The effluent was monitored by UV absorbance detection at 235 nm. The method showed good accuracy, precision and linearity in the concentration range 5–1200 ng/ml. The limit of quantitation was 5 ng/ml, with a precision (C.V.) of 8.91%, which is the same as that achieved by other authors with a previously published GC-MS method. The procedure described in this paper is simple and allows the routine assessment of ticlopidine plasma concentration in pharmacokinetic studies following therapeutic doses in human subjects.     

Determination of ketoconazole in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C(18) column (50 x 3 mm, 5 microm) using acetonitrile-water-formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS-MS detection was by monitoring 531.2-->82.1 (m/z) for ketoconazole, and 733.5-->460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of > or =0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of < or =4.4% (n=6) and the relative errors (REs) from -0.6 to 1.4% for intra-day assay, and < or =8.6% RSD (n=18) and -1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze-thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately -20 degrees C for up to 2 months did not show substantial effects.     

Liquid chromatographic method for the determination of rosiglitazone in human plasma

A robust, accurate and sensitive high-performance liquid chromatographic method for the determination of rosiglitazone (I) in human plasma has been developed. Pioglitazone (II) was used as internal standard. Both I and II are extracted from plasma using a liquid-liquid extraction procedure. Isocratic separation of I and II is carried out using a reversed-phase Zorbax SB C(18), 15-cm column with mobile phase consisting of methanol and a mixed phosphate buffer (10 mM monobasic sodium phosphate and dibasic sodium phosphate, pH adjusted to 2.6 with ortho-phosphoric acid) in the ratio 30:70 (v/v) and quantified by UV detection at 245 nm. Linearity was established over the range 5-1250 ng/ml using 1 ml human plasma. The method is specific, the endogenous components in plasma do not interfere with I and II. C.V. (%) of intra-day samples is less than 5.0% at four concentrations tested namely 5, 10, 500 and 1000 ng/ml. Similarly, over the same nominal concentrations, the precision of inter-day (5 days) samples also results in C.V. (%) less than 5.0%. The recoveries of I and II from human plasma were about 79 and 60%, respectively. This method can be used for routine clinical monitoring of I.     

An analytical method with a single extraction procedure and two separate high performance liquid chromatographic systems for the determination of artesunate, dihydroartemisinin and mefloquine in human plasma for application in clinical pharmacological studies of the drug combination

The combination of two sensitive, selective and reproducible reversed phase liquid chromatographic (RP-HPLC) methods was developed for the determination of artesunate (AS), its active metabolite dihydroartemisinin (DHA) and mefloquine (MQ) in human plasma. Solid phase extraction (SPE) of the plasma samples was carried out on Supelclean LC-18 extraction cartridges. Chromatographic separation of AS, DHA and the internal standard, artemisinin (QHS) was obtained on a Hypersil C4 column with mobile phase consisting of acetonitrile-0.05 M acetic acid adjusted to pH 5.2 with 1.0M NaOH (42:58, v/v) at the flow rate of 1.50 ml/min. The analytes were detected using an electrochemical detector operating in the reductive mode. Chromatography of MQ and the internal standard, chlorpromazine hydrochloride (CPM) was carried out on an Inertsil C8-3 column using methanol-acetonitrile-0.05 M potassium dihydrogen phosphate adjusted to pH 3.9 with 0.5% orthophosphoric acid (50:8:42, v/v/v) at a flow rate of 1.00 ml/min with ultraviolet detection at 284 nm. The mean recoveries of AS and DHA over a concentration range of 30-750 ng/0.5 ml plasma and MQ over a concentration of 75-1500 ng/0.5 ml plasma were above 80% and the accuracy ranged from 91.1 to 103.5%. The within-day coefficients of variation were 1.0-1.4% for AS, 0.4-3.4% for DHA and 0.7-1.5% for MQ. The day-to-day coefficients of variation were 1.3-7.6%, 1.8-7.8% and 2.0-3.4%, respectively. Both the lower limit of quantifications for AS and DHA were at 10 ng/0.5 ml and the lower limit of quantification for MQ was at 25 ng/0.5 ml. The limit of detections were 4 ng/0.5 ml for AS and DHA and 15 ng/0.5 ml for MQ. The method was found to be suitable for use in clinical pharmacological studies.     

High performance liquid chromatographic method for the determination of cetirizine and ambroxol in human plasma and urine--a boxcar approach

A column switching high performance liquid chromatographic method with estimable sensitivity and accuracy was developed for the determination of cetirizine and ambroxol in human plasma using nebivolol as the internal standard. Plasma samples were prepared by liquid-liquid extraction in methylene chloride and a mixture of diethylether (80:20, v/v). The extracted samples were injected into a multifunctional clean-up column Supelcosil LCABZ (50 mm × 4.6 mm, 5 μm particle size) using mobile phase 1 comprising acetonitrile-phosphate buffer (pH 3.5; 20 mM) (20:80, v/v). The eluate of cetirizine and ambroxol were separated to an analytical Kromasil C(8) micro bore column (50 mm × 0.3 mm, 5 μm particle size) via a column switching device. A Kromasil C(18) analytical column (250 mm × 2.1 mm, 5 μm particle size) was used as a separation column. Mobile phase 2 consisting acetonitrile-triethylamine (0.5%) in phosphate buffer (pH 3.5; 20mM) (55:45, v/v) was used for the compound elution. The eluents were detected at 230 nm with photodiode array detector. An aliquot of 150 μl of plasma sample was introduced into the pretreatment column via the auto sampler using mobile phase 1 at a flow rate of 0.5 ml/min, column switching valve being positioned at A. The pretreatment column retained cetirizine, ambroxol and nebivolol (IS) in the column leaving the residual proteins of plasma eluted in void volume and drained out. The switching valve was shifted to position B at 7.5 min. Cetirizine, ambroxol and IS were eluted from the pretreatment column between 7. 5 and 11.5 min and introduced to the concentration column. Finally, cetirizine, ambroxol and IS were introduced to the separation column by switching valve using mobile phase 2 at a flow rate of 0.4 ml/min. During the analysis the pretreatment column was washed for the next analysis and resume to the position A. The total run time was 25 min for a sample. The procedure was repeated for urine analysis also. The method was linear from 2 to 450 ng/ml and 7-300 ng/ml for cetirizine and ambroxol respectively in plasma and 1-500 ng/ml and 5-400 ng/ml, respectively for cetirizine and ambroxol in urine. Intra-day and inter-day precision of cetirizine and ambroxol was below 15% in terms of coefficient of variation and accuracy of cetirizine and ambroxol was ranged from 94 to 101.6% and 91.1 to 100.2%, respectively. The method demonstrated high sensitivity and selectivity and therefore, applied to evaluate pharmacokinetics of cetirizine and ambroxol in healthy human volunteer after a single oral administration. Urine samples obtained from healthy human volunteers and clinical subjects with renal impairment have also been analyzed by the method to compare the elimination pattern. The method was precise and accurate for the estimation of cetirizine and ambroxol both in blood and in urine.     

Sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of stavudine in human plasma

A sensitive method for the determination of stavudine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak Vac, 100 mg, tC(18) solid-phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery C(18), 5 microm, 150 x 2 mm column with a mobile phase consisting of ammonium acetate (0.01 M)-acetonitrile-methanol (800:100:100, v/v/v) at a flow-rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for stavudine was 94% with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been described.