Stereospecific high-performance liquid chromatographic assay of isosakuranetin in rat urine

A stereospecific method of analysis of racemic isosakuranetin (5,7-dihydroxy-4'-methoxyflavanone) in biological fluids is necessary to study pharmacokinetics. A simple high-performance liquid chromatographic method was developed for the determination of isosakuranetin enantiomers. Separation was achieved on a Chiralpak AD-RH column with ultraviolet (UV)-detection at 286 nm. The standard curves in urine were linear ranging from 0.5 to 100.0 microg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV) and was within 12% at the limit of quantitation (0.5 microg/ml). Bias of the assay was <15% and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of isosakuranetin enantiomers in rat urine.     

High-performance liquid chromatography method for the quantification of pantoprazole in human plasma

A sensitive and selective HPLC method with UV detection (290 nm) was developed and validated for quantitation of pantoprazole, proton-pump inhibitor, in human plasma. Following a single-step liquid-liquid extraction with methyl tert-butyl ether/diethyl ether (70/30, v/v), the analyte and internal standard (zonisamide) were separated using an isocratic mobile phase of 10mM phosphate buffer (pH 6.0)/acetonitrile (61/39, v/v) on reverse phase Waters symmetry C18 column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 4%. A linear range of 20-5000 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 1.3-3.2% and 0.7-3.3%, respectively. The between-batch and within-batch bias was -0.5 to 8.2 % and -2.5 to 12.1%, respectively. This validated method is sensitive and repeatable enough to be used in pharmacokinetic studies.     

Determination of alendronate in human urine as 9-fluorenylmethyl derivative by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of alendronate as the 9-fluorenylmethyl derivative (FMOC) in human urine is presented. The sample preparation involved coprecipitation with calcium phosphate, separation on diethylamine (DEA) solid-phase extraction (SPE) cartridge and derivatization with 9-fluorenylmethyl chloroformate in citrate buffer pH 11.9. Liquid chromatography was performed on an octadecylsilica column (150 x 4.6 mm, 3 microm particles); a gradient method with starting mobile phase acetonitrile-methanol-citrate/pyrophosphate buffer (20:15:65 v/v) was employed. The total run time was 21 min. The fluorimetric detector was operated at the following wavelengths: 260 nm (excitation) and 310 nm (emission). Pamdronate was used as the internal standard. The limit of quantitation was 3.5 ng/ml using 5 ml of urine. Within-day and between-day precision expressed by relative standard deviation was less than 8% and inaccuracy did not exceed 9%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of finasteride in human plasma by liquid–liquid extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of finasteride in human plasma is presented. The method is based on liquid–liquid extraction with hexane–isoamylalcohol (98:2, v/v) and reversed-phase chromatography with spectrophotometric detection at 210 nm. The mobile phase consists of acetonitrile–15 mM potassium dihydrogenphosphate (40:60, v/v). Clobazam is used as the internal standard. The limit of quantitation is 4 ng/ml and the calibration curve is linear up to 300 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Quantitation of Valdecoxib in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (210 nm) was developed and validated for quantitation of Valdecoxib in human plasma, the newest addition to the group of non-steroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. The analyte and an internal standard (Rofecoxib) were extracted with diethyl ether/dichloromethane (70/30 (v/v)). The chromatographic separation was performed on reverse phase ODS-AQ column with an isocratic mobile phase of water/methanol (47/53 (v/v)). The lower limit of quantitation was 10 ng/ml, with a relative standard deviation of <20%. A linear range of 10-500 ng/ml was established. This HPLC method was validated with between-batch and within-batch precision of 1.27-7.45 and 0.79-6.12%, respectively. The between-batch and within-batch bias was 0.74-7.40 and -0.93 to 7.70%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Valdecoxib in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is suitable for bioequivalence studies following single dose in healthy volunteers.     

Liquid chromatographic method for the determination of ganciclovir and/or acyclovir in human plasma using pulsed amperometric detection

A rapid high-performance liquid chromatographic method for the quantitation of pseudoephedrine in human plasma is presented. The sample preparation involved liquid-liquid extraction of pseudoephedrine from alkalised plasma with hexane-isoamylalcohol (9:1, v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on an octadecylsilica column (50 x 4 mm, 5 microm particles); the mobile phase consisted of acetonitrile-phosphate buffer containing 0.1% of triethylamine, pH 2.4 (5:95, v/v). The run time was 4 min. The spectrophotometric detector was operated at 195 nm. Codeine was used as the internal standard. The limit of quantitation was 5.8 ng/ml using 0.5 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Validated liquid chromatographic ultraviolet method for the quantitation of Etoricoxib in human plasma using liquid-liquid extraction

A simple, sensitive and specific HPLC method with UV detection (284 nm) was developed and validated for quantitation of Etoricoxib in human plasma, the newest addition to the group of nonsteroidal anti-inflammatory drugs-a highly selective cyclooxygenase-2 inhibitor. Following a single-step liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v), the analyte and internal standard (Zaleplon) were separated using an isocratic mobile phase of water/acetonitrile (58/42, v/v) on reverse phase Waters symmetry C(18) column. The lower limit of quantitation was 5 ng/mL, with a relative standard deviation of less than 20%. A linear range of 5-2500 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 4.1-5.1% and 1.1-2.4%, respectively. The between- and within-batch bias was -3.8-4.7% and -0.6-9.4%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of Etoricoxib in plasma was >90%, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive and simple with between-batch precision of <6% and was used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of entacapone in human plasma

A simple, sensitive and selective HPLC method with UV detection (315 nm) was developed and validated for quantitation of entacapone in human plasma, the newest addition to the group of antiparkinsonian agents. Following a single-step liquid-liquid extraction (LLE) with ethyl acetate/n-hexane (30/70, v/v), the analyte and internal standard (rofecoxib) were separated using an isocratic mobile phase of 30 mM phosphate buffer (pH 2.75)/acetonitrile (62/38, v/v) on a reverse phase C18 column. The lower limit of quantitation was 25 ng/mL, with a relative standard deviation of less than 8%. A linear range of 25-2500 ng/mL was established. This HPLC method was validated with between-batch and within-batch precision of 2.2-4.2% and 1.7-7.8%, respectively. The between-batch and within-batch accuracy was 98.7-107.5% and 97.5-106.0%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of entacapone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

High-performance liquid chromatography method for the quantification of rabeprazole in human plasma using solid-phase extraction

A simple, sensitive and selective HPLC method with UV detection (284 nm) was developed and validated for quantitation of rabeprazole in human plasma, the newest addition to the group of proton-pump inhibitors. Following solid-phase extraction using Waters Oasistrade mark SPE cartridges, the analyte and internal standard (Pantoprazole) were separated using an isocratic mobile phase of 5 mM ammonium acetate buffer (pH adjusted to 7.4 with sodium hydroxide solution)/acetonitrile/methanol (45/20/35, v/v) on reverse phase Waters symmetry C(18) column. The lower limit of quantitation was 20 ng/mL, with a relative standard deviation of less than 8%. A linear range of 20-1000 ng/mL was established. This HPLC method was validated with between- and within-batch precision of 2.4-7.2% and 2.2-7.3%, respectively. The between- and within-batch bias was -1.7 to 2.6% and -2.6 to 2.1%, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of rabeprazole in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 3 months storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester

A stereospecific HPLC method for the quantitation of CGP 49309 in samples of its corresponding enantiomer valsartan has been developed and validated. The enantiomeric separation was achieved on a 5 μm silica-bonded α₁-acid glycoprotein column (Chiral AGP) with a phosphate buffer, pH 7, containing 2% (v/v) 2-propanol as a mobile phase. The linearity was established in the range 0.1–4% (r>;0.999). The limit of quantitation was 0.1% and the limit of detection was 0.04%. The accuracy of the method was found to be 96.7% (average). For the precision (repeatability), a relative standard deviation value of 2.4% was found. Similarly, a stereoselective HPLC method was also developed and validated for the quantitation of the enantiomer of the starting material used for the synthesis of valsartan, namely (R)-valinebenzyl ester tosylate. Baseline resolution of the enantiomers of valinebenzyl ester tosylate could be achieved on the chiral crown ether column Crownpak CR (Daicel) at 50°C using water-methanol-trifluoroacetic acid (850:150:1, v/v) as a mobile phase. The linearity was established in the range 0.5-5% (r>;0.999). The accuracy of the method was found to be 100.5% (average). For the precision (repeatability), a relative standard deviation value of 3.4% was found. Both methods were found to be suitable for the analysis of the respective analytes.     

High performance liquid chromatographic method for the determination of sumatriptan with fluorescence detection in human plasma

A rapid and sensitive high performance liquid chromatography (HPLC) method with fluorescence detection has been developed for the determination of sumatriptan in human plasma. The procedure involved a liquid-liquid extraction of sumatriptan and terazosin (internal standard) from human plasma with ethyl acetate. Chromatography was performed by isocratic reverse phase separation on a C18 column. Fluorescence detection was achieved with an excitation wavelength of 225 nm and an emission wavelength of 350 nm. The standard curve was linear over a working range of 1-100 ng/ml and gave an average correlation coefficient of 0.9997 during validation. The limit of quantitation (LOQ) of this method was 1 ng/ml. The absolute recovery was 92.6% for sumatriptan and 95.6% for the internal standard. The inter-day and intra-day precision and accuracy were between 0.8-3.3 and 1.1-6.3%, respectively. This method is simple, sensitive and suitable for pharmacokinetics or bioequivalence studies.     

Quantitative HPLC-UV method for the determination of firocoxib from horse and dog plasma

A sensitive reversed-phase HPLC-UV method was developed for the determination of firocoxib, a novel and highly selective COX-2 inhibitor, in plasma. A 1.0 mL dog or horse plasma sample is mixed with water and passed through a hydrophobic-lipophilic copolymer solid-phase extraction column to isolate firocoxib. Quantitation is based on an external standard curve. The method has a validated limit of quantitation of 25 ng/mL and a limit of detection of 10 ng/mL. The validated upper limit of quantitation was 2500 ng/mL for horses and 10,000 ng/mL for dogs. The average recoveries ranged from 88-93% for horse plasma and 96-103% for dog plasma. The coefficient of variation in all cases was less than 10%. This method is suitable for the analysis of clinical samples from pharmacokinetic and bioequivalence studies and drug monitoring.     

Determination of huperzine A in formulated products by reversed-phase-liquid chromatography using diode array and electrospray ionization mass spectrometric detection.

A precise and selective high-performance liquid chromatographic (HPLC) method with diode-array detection for quantifying huperzine A in formulated products was developed and validated. A liquid chromatographic-mass spectrometric (LC/MS) procedure was devised to confirm the HPLC method. Huperzine A was dissolved in 1,2-dichloroethane, chromatographed on a YMCBasic C18 column, and detected at 308 nm. A gradient mobile phase of 10 mM ammonium acetate (pH = 3.5)--methanol was used. Identification was based on retention time, UV spectra and mass spectra by comparison with a commercial standard. The UV peak areas were used for quantitation of huperzine A content. The correlation coefficient (R2) of the calibration curve was 1 over the range 0.8-11.6 microg/ml. Overall recovery of huperzine A was 103.9% +/- 1.8 (mean +/- SD). Relative standard deviations for intra- and interday precision were < 2%.     

Simple and sensitive method for determination of metronidazole in human serum by high-performance liquid chromatography

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml⁻¹ using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile–aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 μg ml⁻¹. Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 μg ml⁻¹ and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.     

High-performance liquid chromatography-mass spectrometry method for the determination of paroxetine in human plasma

A rapid and specific liquid chromatographic mass spectrometric (LC-MS-MS) method has been developed for the determination of paroxetine in human plasma. The procedure involves a liquid-liquid extraction of paroxetine and fluoxetine (internal standard) with cyclohexane-ethyl acetate. The standard curve was linear over a working range of 0.2-50 ng/ml. The lower limit of quantitation was 0.2 ng/ml. No endogenous compounds were found to interfere with the analysis. The absolute recovery was 70.8% for paroxetine and 84.1% for the internal standard. The accuracy of inter-assay and intra-assay accuracy was in the ranges -4.8 to -0.5% and -3.4 to 4.8%, respectively. This method proved to be suitable for bioequivalence studies by being simple, selective and reproducible.     

Chiral reversed phase high-performance liquid chromatography for determining propranolol enantiomers in transgenic Chinese hamster CHL cell lines expressing human cytochrome P450

An enantioselective assay for S-(-)- and R-(+)-propranolol in transgenic Chinese hamster CHL cell lines, expressing human cytochrome P450 (CYP), was developed. The method involves extraction of propranolol from the S(9) incubates, using S-(+)-propafenone as internal standard, chiral derivatization with 2,3,4,6-tetra-O-beta-D-glucopranosyl isothiocyanate and quantitation by reversed phase high-performance liquid chromatography system with UV detection (lambda=220 nm). A baseline separation of propranolol enantiomers was achieved on a 5-microm reverse-phase ODS column, with a mixture of methanol/water/glacial acetic acid (67:33:0.05, v/v) as mobile phase. The assay is linear from 5 to 500 microM for each enantiomer. The analytical method affords average recoveries of 99.2% and 98.8% for S-(-)- and R-(+)-propranolol, respectively. The limit of quantitation for the method is 5 microM for both S-(-)- and R-(+)-propranolol. The reproducibility of the assay is satisfactory (RSD < 10%). The method allowed study of the depletion of S-(-)- and R-(+)-propranolol in transgenic Chinese hamster CHL cell lines expressing CYP3A4, CYP2C18 and CYP2C9.     

A simple and sensitive HPLC method for determination of gliclazide in human serum

A simple, rapid and specific method for analysis of gliclazide in serum by a sensitive high-performance liquid chromatographic method is described. Only 100 microl of serum and a little sample work-up is required. A simple procedure of extraction by toluene followed by evaporation to dryness under a gentle stream of air and dissolving the dried residue in mobile was used. The gliclazide peak was separated from endogenous peaks on a C(8) column by a mobile phase of acetonitrile-water (45:55, v/v), pH 3. Gliclazide and internal standard (phenytoin) were eluted at 6.8 and 3.8 min, respectively. The limit of quantitation (LOQ) for gliclazide in serum was 75 ng/ml at 230 nm. The method was linear over the range of 75-10,000 ng/ml with r(2) of 0.999. Mean recovery for gliclazide and internal standard was 84.5 and 87%, respectively.     

A novel chemiluminescence method for the determination of orciprenaline based on ferricyanide-rhodamine 6G.

A novel flow injection chemiluminescence method for the determination of orciprenaline was developed. The method is based on the chemiluminescence (CL) reaction of orciprenaline with potassium ferricyanide in sodium hydroxide medium, sensitized by the fluorescent dye rhodamine 6G. The proposed procedure allows quantitation of orciprenaline in the concentration range 0.01-1.2 microg/mL, with a detection limit of 7.2 x 10(-3) microg/mL. The relative standard deviation (RSD) is 2.7% for 0.1 microg/mL orciprenaline (n = 9). The sampling frequency was calculated at approximately 120/h. The method was successfully applied to the determination of orciprenaline in pharmaceutical preparations. A brief discussion on the possible CL reaction mechanism is presented.     

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.     

Stereospecific analysis of sakuranetin by high-performance liquid chromatography: pharmacokinetic and botanical applications

A stereospecific method for analysis of sakuranetin was developed. Separation was accomplished using a Chiralpak AD-RH column with UV (ultraviolet) detection at 288 nm. The stereospecific linear calibration curves ranged from 0.5 to 100 microg/mL. The mean extraction efficiency was >98%. Precision of the assay was <12% (relative standard deviation (R.S.D.)%), and within 10% at the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 10%, and within 5% at the limit of quantitation. The assay was applied successfully to pharmacokinetic quantification in rats, and the stereospecific quantification in oranges, grapefruit juice, and matico (Piper aduncum L.).