Determination in serum of some barbiturates using micellar liquid chromatography with direct injection

A procedure was developed for the determination of several barbiturates, amobarbital, barbital, hexobarbital, and secobarbital, using a C18 column (120 x 4.6mm) and micellar liquid chromatography (MLC) mobile phases containing sodium dodecyl sulfate (SDS) and propanol, butanol, or pentanol as a modifier, with UV detection at 230nm. After the application of an interpretative strategy of optimization, the four barbiturates can be resolved and determined in serum samples, allowing the direct injection in 0.10M SDS-4% (v/v) butanol, pH 7, with an analysis time below 8 min. In the proposed MLC procedure, linearities (r >0.999), limits of detection (ngmL(-1)) in the 30-70 range, repeatabilities, and intermediate precision below 1.8% are adequate for the quantification. The proposed method could be applied to the determination of barbiturates in serum samples with recoveries that agreed with the concentration added.     

Rapid and sensitive determination of nicotine in formulations and biological fluid using micellar liquid chromatography with electrochemical detection

Nicotine can be determined in pharmaceuticals and biological fluids by micellar liquid chromatography (MLC) using a C18 column, a mobile phase containing sodium dodecyl sulphate 0.15 M–6% (v/v) pentanol–NaH₂PO₄ 0.01 M (pH 6)–KCl 0.001 M, with electrochemical detection at 0.8 V. In the optimization step, the influence of the modifiers propanol, butanol and pentanol, and the voltage has been studied. With the proposed method the analysis time is below than 8 min, linearity better than 0.999, limits of detection and quantification (ng/ml) was 4 and 12 respectively, repeatability and intermediate precision below 1.8%, and all these parameters are adequate for the quantification of nicotine in chewing gum, dermal patches, tobacco and serum samples either by a pharmacologist, pathologist or toxicologist.     

Two liquid chromatographic approaches for the simultaneous determination of xipamide and its degradation product (2,6‐xylidine) using time‐programmed fluorescence detection

A study of the performance of reversed‐phase chromatography with a programmable multiwavelength fluorimetric technique using either conventional hydro‐organic or micellar eluent is established for the determination of xipamide (XIP) in the presence of its degradation product, 2,6‐xylidine (XY). In conventional liquid chromatography (CLC), the analyses were carried out on a Promosil ODS 100 Å column (250 mm × 4.6 mm i.d., 5 μm) using a mobile phase consisting of methanol/0.1 M phosphate buffer (65: 35, v/v) at pH 4.0. For micellar liquid chromatography (MLC), a short Spherisorb column (150 mm × 4.6 mm i.d., 5 μm) was employed in conjunction with a greener mobile phase (pH 5.0) containing 0.1 M sodium dodecyl sulfate and 15% n‐propanol. CLC proved to be superior to MLC in terms of sensitivity for the determination of the degradation product because it could detect trace amounts down to 10.0 ng/ml of XY as a degradation product in XIP. However, MLC represents an eco‐friendly approach for the simultaneous determination of XIP and XY. In addition, the opportunity for the direct introduction of biological matrices into the chromatographic system is one of the distinctive benefits of MLC. The proposed methods were applied for the determination of XIP in its tablets, human urine and content uniformity testing. The results of the proposed methods were statistically compared with those obtained using the comparison fluorimetric method, revealing no significant differences in the performance of the methods regarding accuracy and precision.     

Direct determination of verapamil in urine and serum samples by micellar liquid chromatography and fluorescence detection

Verapamil, a calcium channel antagonist, is one of the most commonly prescribed drugs in the treatment of hypertension. In this work, it was determined in serum and urine samples by a sensitive and precise chromatographic procedure without any pre-treatment step in a C18 column using a micellar mobile phase of 0.15M sodium dodecyl sulfate and 5% pentanol at pH 7. Fluorescence detection set at 230 nm (excitation) and 312 nm (emission) was used. Verapamil is eluted at 12.5 min with no interference by the protein band or endogenous compounds. Linearities (r > 0.998), as well as intra- and inter-day precision, were studied in the validation of the method. LODs were also calculated to be 11.0, 18.5 and 20.2 ng/mL in micellar solution, serum and urine, respectively. Recoveries in the biological matrices were in the 97-99% range. Drug excretion in urine was studied in a volunteer receiving treatment for hypertension, and verapamil, as an unchanged drug, was separated from other metabolites. The procedure developed can be useful in the field of toxicology and clinical analysis.     

Chromatographic performance of large-pore versus small-pore columns in micellar liquid chromatography

Micellar liquid chromatography (MLC) is useful in bioanalysis because proteinaceous biofluids can be directly injected onto the column. The technique has been limited in part because of the apparently weak eluting power of micellar mobile phases. It has recently been shown [Anal. Chem. 72 (2000) 294] that this may be overcome by the use of large pore size stationary phases. In this work, large-pore (1000 A) C(18) stationary phases were evaluated relative to conventional small-pore (100 A) C(18) stationary phases for the direct sample injection of drugs in plasma. Furthermore, the difference between the large and small pore phases in gradient elution separations of mixtures of widely varying hydrophobicities was investigated. Large-pore stationary phases were found to be very effective for eluting moderately to highly hydrophobic compounds such as ibuprofen, crotamiton, propranolol, and dodecanophenone, which were highly retained on the small-pore stationary phases typically used in MLC. The advantages of direct introduction of biological samples (drugs in plasma) and rapid column re-equilibration after gradient elution in MLC were maintained with large-pore phases. Finally, recoveries, precision, linearity, and detection limits for the determination of quinidine and DPC 961 in spiked bovine plasma were somewhat better using MLC with wide pore phases.     

High-performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum: Application to a bioequivalence study

A sensitive and rapid method is described for determination of clopidogrel carboxylic acid (CCA), the inactive metabolite of anti platelet agent, clopidogrel, in human serum. The analytical procedure involves liquid-liquid extraction of the analyte and an internal standard (phenytoin) with ethyl acetate. A mobile phase consisting of 0.05 M phosphate buffer containing triethylamine (0.5 mL/L; pH 5.7) and acetonitrile (56:44 v/v) was used and chromatographic separation was achieved using C18 analytical column at detector wavelength of 220 nm. The calibration curves were linear over a concentration range of 0.05-10 microg/mL of CCA in human serum. The total run time of analysis was 5.5 min and the lower limits of detection (LOD) and quantification (LOQ) were 0.02 and 0.05 microg/mL, respectively. The method validation was carried out in terms of specificity, sensitivity, linearity, precision, accuracy and stability. The validated method was applied in a randomized cross-over bioequivalence study of two different clopidogrel preparations in 24 healthy volunteers.     

Determination of vertilmicin in rat serum by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

A procedure for the high-performance liquid chromatographic determination of vertilmicin in rat serum was described using pre-column derivatization. The serum proteins were precipitated with acetonitrile and vertilmicin in the supernatant was derivatized with 1-fluoro-2,4-dinitrobenzene. Etimicin was selected as the internal standard. The mobile phase consisted of methanol--20mM ammonium acetate (80:20, v/v), and flow-rate was 0.9 ml/min. Ultraviolet detection was set at 365 nm. The reaction products were chromatographed on a C(18) column kept at 40 degrees C. A good linearity was found in the range of 0.5-250 microg/ml. Both intra- and inter-day precisions of vertilmicin, expressed as the relative standard deviation, were less than 7.4%. Accuracy, expressed as the relative error, ranged from -0.1 to 3.6%. The mean absolute recovery of vertilmicin at three different concentrations was 92.5%. Serum volumes of 50 microl were sufficient for the determination of vertilmicin. The method was proved suitable for the pharmacokinetic study of vertilmicin in rats.     

Improvement and validation of a liquid chromatographic method for the determination of levosulpiride in human serum and urine

A rapid, selective and highly sensitive reversed-phase high-performance liquid chromatography (HPLC) method was developed for the determination of levosulpiride, 5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy benzamide, in human serum and urine. The method involved the extraction with a dichloromethane followed by back-extraction into 0.025 M sulfuric acid. HPLC analysis was carried out using reversed-phase isocratic elution with a Luna C(18)(2) 5 microm column, a mobile phase of acetonitrile-0.01 M potassium hydrogen phosphate (30:70, v/v, adjusted to pH 8.5 with triethylamine), and a fluorescence detector with excitation at 300 nm and emission at 365 nm. The chromatograms showed good resolution and sensitivity and no interference of human serum and urine. The calibration curves were linear over the concentration range 0.25-200 ng/ml for serum and 0.2-20 microg/ml for urine with correlation coefficients greater than 0.997. Intra- and inter-day assay precision and accuracy fulfilled the international requirements. The mean absolute recovery for human serum was 89.8+/-3.7%. The lower limits of quantitation in human serum and urine were 0.25 ng/ml and 0.2 microg/ml, respectively, which were sensitive enough for pharmacokinetic studies. Stability studies showed that levosulpiride in human serum and urine was stable during storage, or during the assay procedure. This method was successfully applied to the study of pharmacokinetics of levosulpiride in human volunteers following a single oral administration of levosulpiride (25 mg) tablet.     

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Method development and validation for the simultaneous determination of imatinib mesylate and N-desmethyl imatinib using rapid resolution high performance liquid chromatography coupled with UV-detection

We developed a simple and sensitive method for the simultaneous detection of imatinib mesylate (IM) and its active metabolite, N-desmethyl imatinib (M1), in human serum samples. Separation was successfully achieved using an Agilent(?) ZORBAX Eclipse plus C(18) reversed phase column (50 mm × 2.1 mm, i.d.; 1.8 μm) under isocratic mobile phase conditions consisting of acetonitrile: 0.02 M potassium dihydrogen phosphate with 0.2% triethylamine at pH 3 (25:75, v/v) and ultra-violet detection was achieved at 235 nm. Extraction of the target compounds was completed using 100% cold acetonitrile. Good linearities (r(2)>0.99) for both IM and M1 were achieved for the concentration ranges of 50-1800 ng/mL and 50-360 ng/mL, respectively. The detection limits were 20 ng/mL and 10 ng/mL for M1 and IM, respectively. The intra- and inter-day precisions were less than 1% with percent recoveries of more than 90%. The method was successfully applied to calculate the pharmacokinetic parameters of chronic myeloid leukemia patients receiving imatinib. The method is suitable to be routinely applied for determination of IM and M1 in serum.     

Determination of echinacoside in rat serum by reversed-phase high-performance liquid chromatography with ultraviolet detection and its application to pharmacokinetics and bioavailability

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of echinacoside (ECH) in rat serum. After protein precipitation of serum sample with trichloroacetic acid, the supernatant was directly injected and analyzed on a C(18) CapcellACR analytical column (150 mm x 4.6mm I.D. 5 microm) with a mobile phase consisting of acetonitrile-0.5% acetic acid (15.5:84.5, v/v). The UV detector was set at 330 nm. The lower limit of detection and quantification were 9 and 29.2 ng/mL, respectively, and the calibration curves were linear over the concentration range of 29.2-18250 ng/mL. The assay method was successfully applied to the study of the pharmacokinetics and bioavailability of ECH in rat.     

Determination of atorvastatin in human serum by reversed-phase high-performance liquid chromatography with UV detection

A rapid and sensitive high-performance liquid chromatographic method was validated and described for determination of atorvastatin in human serum. Following liquid-liquid extraction of the drug and an internal standard (sodium diclofenac), chromatographic separation was accomplished using C18 analytical column with a mobile phase consisting of sodium phosphate buffer (0.05 M, pH 4.0) and methanol (33:67, v/v). Atorvastatin and the internal standard were detected by ultraviolet absorbance at 247 nm. The average recoveries of the drug and internal standard were 95 and 80%, respectively. The lower limits of detection and quantification were 1 and 4 ng/ml, respectively, and the calibration curves were linear over a concentration range of 4-256 ng/ml of atorvastatin in human serum. The analysis performance was studied and the method was applied in a randomized cross-over bioequivalence study of two different atorvastatin preparations in 12 healthy volunteers.     

Improvement and validation of an HPLC method for examining the effects of the MDR1 gene polymorphism on sparfloxacin pharmacokinetics

A rapid, simple, accurate, and precise reverse-phase high-performance liquid chromatography (HPLC) method for measuring sparfloxacin in human serum was improved, validated, and applied to determine the influence of polymorphisms in MDR1 (exons 12, 21, and 26) gene on sparfloxacin pharmacokinetics. Sparfloxacin and an internal standard, ciprofloxacin, were extracted from human serum by protein precipitation with dilution and analyzed on a Luna C(18) 5-microm column in a mobile phase of acetonitrile-0.035 M perchloric acid (28:72, v/v, adjusted to pH 2.0 with 0.015 M triethylamine) and UV detection at 300 nm. This analysis was performed at three different laboratories using the same quality control (QC) samples. The chromatograms showed good resolution, sensitivity, and no interference by human serum. The method showed linear responses over a concentration range of 0.05-2 microg/ml, with correlation coefficients of greater than 0.999 at the three laboratories. Intra- and inter-day assay precision and accuracy fulfilled international requirements. The mean absolute recovery for human serum was 98.8+/-5.7%. Sparfloxacin in human serum was stable during storage and the assay procedure. The lower limit of quantification using 0.2 ml of serum was 0.05 microg/ml, which was sensitive enough for pharmacokinetic studies. This method was used to study the pharmacokinetics of sparfloxacin in human volunteers, following a single oral administration of sparfloxacin (100 mg) two tablets at three different laboratories. MDR1 polymorphisms did not significantly (P < 0.01) affect the pharmacokinetic parameters (AUC and C(max)) of sparfloxacin.     

Solid-phase extraction of methadone enantiomers and benzodiazepines in biological fluids by two polymeric cartridges for liquid chromatographic analysis

The aim of this work was to present the advantages of two polymeric cartridges (Oasis HLB from Waters and Abselut Nexus from Varian) for the solid-phase extraction of methadone enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and of some benzodiazepines (diazepam, flunitrazepam, nitrazepam, oxazepam) in serum and urine in comparison with classical C18-bonded-silica cartridges or liquid extraction. After addition of serum or urine samples, these two cartridges were washed with a water-methanol mixture (95:5, v/v) and eluted with diethylether. After rapid evaporation, the residue was regenerated with mobile phase and injected either in a chiral column (Cyclobond I-2000 RSP) for methadone enantiomers and its metabolite or in a reversed-phase column (Symmetry Shield RP8) for benzodiazepines. The results showed that the chromatograms of blank serum and urine were cleaner than those obtained from classical solid-phase extraction or liquid extraction. The recoveries from these two polymeric cartridges were higher (95-102%) than those obtained by the two previous classical methods and the total time for extraction and solvent evaporation was also shorter (about 6-7 min). For methadone and benzodiazepine extraction, the use of acidic or alkaline buffer was not necessary.     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

Development of a liquid chromatography-mass spectrometry method for monitoring the angiotensin-converting enzyme inhibitor lisinopril in serum

In this study, a sensitive, specific, precise and accurate method for lisonopril quantitative determination in human serum was developed and validated. The method comprises lisinopril isolation from serum by means of solid-phase extraction followed by its quantification by liquid chromatography-mass spectrometry. Chromatographic separation was performed at 55 degrees C on Kromasil C(18) 5 micrometer 250x3.2 mm HPLC column with mobile phase composed of 50 mM ammonium formate buffer (pH 3)-acetonirile-methanol (72:7:21, v/v/v). A Finnigan AQA benchtop mass spectrometer with a pneumatically assisted electrospray (ES) interface and a single quadrupole mass filter was used to detect and quantify lisinopril in column effluent. Ion signals were acquired by selected ion monitoring of the protonated lisinopril ion m/z=406.5 (M+1). The detector response was linear with r>0.9993 in the investigated concentration range 6-150 ng/ml. The mean recovery of lisinopril from serum samples was 88%. The limit of quantitation for lisinopril was 6 ng/ml with a signal-to-noise ratio at this concentration level S/N=34.75+/-3.9 (n=4).     

Investigation of chromatographic conditions for the separation of cefuroxime axetil and its geometric isomer

The optimal chromatographic conditions for the separation of the syn- and anti-geometric isomers of cefuroxime axetil applying RP-HPLC and micellar liquid chromatography (MLC) methods were investigated. The possibility to separate diastereoisomers of syn- and anti-cefuroxime axetil was observed. Investigations were performed using three columns, two classical silicas and one with hybrid particle technology. Three aqueous-organic and one micellar mobile phases were used. The best results were achieved using micellar mobile phase. Optimization study was performed using different micellar mobile phases. MLC method is sensitive and applicable in purity and stability testing.     

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.     

Simultaneous determination of doxifluridine and 5-fluorouracil in monkey serum by high performance liquid chromatography with tandem mass spectrometry

A reverse-phase high performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of doxifluridine and its active metabolite 5-fluorouracil in monkey serum. A liquid/liquid extraction with ethyl acetate (90%) and isopropyl alcohol (10%) was used to extract simultaneously doxifluridine and 5-FU which have considerable difference in the polarity. Optimum chromatographic separation was achieved on a Agilent Zorbax C(18) (100 mm x 2.1mm, 3.5 microm) column with a mobile phase of methanol-water (20:80, v/v). The flow rate was 0.2 mL/min with total cycle time of 5 min. The lower limit of quantification (LLOQ) was validated at 10.0 ng/mL of serum for both doxifluridine and 5-FU. Accuracy and precision of quality control (QC) samples for both compounds met FDA Guidance criteria of +/-15% with average QC accuracy of 95.5-105.0% and coefficients of variation of 1.1-9.5% in the 10-2000 ng/mL concentration range. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability to support the analysis of monkey serum samples.     

HPLC determination of pirenzepine dihydrochloride in rabbit aqueous humor

Pirenzepine was considered as a pharmacologic agent of preventing form-deprivation myopia. To assess the ocular bioavailability of pirenzepine, a HPLC method for determination of pirenzepine in rabbit aqueous humor was developed. An HPLC system was used in the reverse phase mode for the determination of pirenzepine. A Luna RP18 5 microm 4.6 mm x 150 mm column was employed at 35 degrees C. The mobile phase was methanol/0.02 M KH2PO4/sodium 1-pentanesulfonate (350/650/1, v/v/m, pH was adjusted to 8.0 by dropping 1M NaOH). The flow rate was 1 ml/min. Pirenzepine was monitored at 280 nm. Sample treatment procedure consists of deproteinisation with methanol. Calibration curves fitted by plotting the peak area versus concentration were linear in the range 20-400 ng/ml. The limit of quantification (LOQ) of present method was 20 ng/ml. Within-day and inter-day coefficient of variation was lower than 10%. Analytical recoveries were determined as 92.4, 95.4 and 101.4% at concentrations of 40, 200 and 400 ng/ml. In conclusion, this HPLC method using a simple sample treatment procedure appears suitable for monitoring ocular concentration of pirenzepine.