Reversed-phase ion-pair liquid chromatographic method for the determination of low concentrations of haloperidol in plasma

A sensitive liquid chromatographic method for the determination of haloperidol in plasma is described. The efficient and simple extraction procedure, followed by reversed-phase ion-pair liquid chromatography on a 3-micron octadecylsilica column and UV absorbance detection, makes it possible to determine concentrations down to 0.5 nmol/l with acceptable precision. In a pharmacokinetic study, in which 5 mg of haloperidol were given orally, the plasma levels were followed for 48 h.     

A fluorimetric, column-switching HPLC and its application to an elimination study of LLU-alpha enantiomers in rat plasma

A method for the enantiomeric determination of 2,7,8-trimethyl-2-(beta-carboxyethyl)-6-hydroxy chroman (LLU-alpha, gamma-CEHC) in rat plasma was developed using high-performance liquid chromatography (HPLC) with a fluorimetric derivatization with 4-N, N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) followed by O-acetylation with acetyl chloride. The proposed HPLC system used two non-chiral columns (phenyl and octadecylsilica) and a chiral column (a modified cellulose type), which were connected via two column-switching valves. A derivatized sample prepared from rat plasma was first separated on the phenyl column, and the fraction including LLU-alpha derivative was introduced to the octadecylsilica column to quantify the concentration of the mixture of S- and R-LLU-alpha. Finally, the LLU-alpha derivative was directly injected into the chiral column to obtain the ratio of the enantiomers. The proposed HPLC system was applied to the enantiomeric determination of LLU-alpha in plasma after intravenous administration of racemic LLU-alpha. S-LLU-alpha was eliminated faster than R-LLU-alpha, and its concentration in plasma decreased to one-third at 2 min after dosing.     

Reversed-phase liquid chromatographic method for the simultaneous determination of the antimalarial drugs sulfadoxine, pyrimethamine, mefloquine and its major carboxylic metabolite in plasma.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of sulfadoxine, pyrimethamine, mefloquine and the carboxylic metabolite of mefloquine in plasma is described. After the proteins have been precipitated with a combination of zinc sulphate and acetonitrile containing two internal standards, pyrimethamine and mefloquine are extracted as bases and sulfadoxine and the carboxylic metabolite of mefloquine as ion-pairs with tetrabutylammonium. The drugs are separated by HPLC on a 3 micron octadecylsilica column with ultraviolet detection at 229 nm. The method is simple and reliable and enables the simultaneous determination of the drugs in 600-microliter plasma samples with a sensitivity suitable for standard drug monitoring purposes.     

Simultaneous determination of D-lactic acid and 3-hydroxybutyric acid in rat plasma using a column-switching HPLC with fluorescent derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ)

A highly sensitive method for the determination of D-lactic acid and 3-hydroxybutyric acid (3-HB) in rat plasma was developed using high-performance liquid chromatography with octadecylsilica (ODS) connected to a chiral column. At first, (D + L)-lactic acid and 3-HB in the plasma were derivatized with a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), separated on the ODS column and determined fluorimetrically at 547 nm with 491 nm of excitation wavelength. During the separation step on the ODS, the peak fraction of (D + L)-lactate derivative was introduced directly to a phenylcarbamoylated beta-cyclodextrin chiral column by changing the flow of the eluent via a six-port valve. Then, D-lactate derivative was separated enantiomerically from the L-lactate derivative, and the enantiomeric ratio was determined from the chromatogram. Intra- and inter-day accuracy values for the determination of D-lactic acid in 10 microL of rat plasma were 97.8-109.2 and 98.4-109.9%, and those for 3-HB were 99.8-108.4 and 99.8-103.8%, respectively. The intra- and inter-day precision values were within 4.6 and 5.1% for D-lactic acid, and 2.7 and 2.4% for 3-HB, respectively. The detection limits for D-lactic acid and 3-HB were approximately 2.0 and 0.04 microM, respectively (signal-to-noise ratio 3). The proposed method was applied to the plasma of diabetic rats induced by intraperitoneal administration of streptozotocin, and the significant increases of both D-lactic acid and 3-HB concentrations were observed in the diabetic rats as compared to the normal rats.     

Determination of remoxipride in plasma and urine by reversed-phase column liquid chromatography

A reversed-phase column liquid chromatographic method for the determination of remoxipride, a novel antipsychotic drug, in biological fluids is described. A simple one-step extraction is used followed by liquid chromatography on a 3-microns octadecylsilica column and ultraviolet absorbance detection. The method is accurate and precise for clinical remoxipride levels in both plasma and urine. For situations where a higher sensitivity is necessary a two-step extraction and a modified mobile phase are used. With this modification plasma concentrations down to 2 nM can be determined with acceptable precision.     

Solid phase extraction and HPLC determination of spiramycin in plasma and vitreous concentrations

A method for extracting spiramycin by an octadecylsilica cartridge is described for plasma or vitreous samples. The macrolide antibiotic is then measured by reversed-phase HPLC with UV detection. The limit of detection is estimated to be 50 ng/mL. The coefficient of variation for the procedure is 6.1% and 5.2% for the range of concentrations 0.2 micrograms/mL and 10 micrograms/mL respectively. By this method, pharmacokinetic profiles were performed for five adult patients. Spiramycin could be accurately measured in the vitreous humour, allowing the determination of antibiotic at its site of action.     

Selective system of identification and determination of antidepressants and neuroleptics in serum or plasma by solid-phase extraction followed by high-performance liquid chromatography with photodiode-array detection in analytical toxicology.

A selective off-line solid-phase isolation of antidepressants, neuroleptics and other structurally related basic drugs from plasma or serum prior to high-performance liquid chromatography was tested and optimized for general use in toxicological analyses where concomitant drugs can be encountered. The sequential elution preseparated drug mixtures and simplified the subsequent analytical steps. High isolation efficiencies of cyano-bonded silica cartridges from Baker for fifteen amine drugs were determined. Isocratic chromatography on octadecylsilica proved to be very suitable for broad practical applications in complicated cases. The identification of an unknown peak was supported by photodiode-array detection in the range 200-400 nm with a resolution of 2 nm. The linearity of the assay from therapeutic to toxic concentrations was attained. Sufficient sensitivities covering low therapeutic levels of parent drugs and their demethylated metabolites were reached. The system is flexible and allows various methods of quantitative assay to be devised according to the conditions of a particular case in clinical or forensic toxicology.     

Liquid chromatography-tandem mass spectrometry for quantification of lacosamide, an antiepileptic drug, in rat plasma and its application to pharmacokinetic study

A rapid, simple and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) was developed for the determination of an antiepileptic drug, lacosamide, in rat plasma. The method involves the addition of acetonitrile and internal standard solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on column packed with octadecylsilica (5 µm, 2.0 × 50 mm) with 0.1% formic acid and acetonitrile as mobile phase, and the detection was performed on tandem mass spectrometry by the multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear over the concentration range from 0.3 to 1000 ng/mL. The lower limit of quantification was 0.3 ng/mL using 50 μL of rat plasma sample. The intra‐ and inter‐assay precision and accuracy were found to be less than 11.7 and 8.8%, respectively. The developed analytical method was successfully applied to the pharmacokinetic study of lacosamide in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantification of lurasidone, an atypical antipsychotic drug, in rat plasma with high-performance liquid chromatography with tandem mass spectrometry

A liquid chromatography–tandem mass spectrometric (LC/MS/MS) method was developed for the determination of an atypical antipsychotic drug, lurasidone, in rat plasma. The method involves the addition of acetonitrile and ziprasidone (internal standard) solution to plasma samples, followed by centrifugation. An aliquot of the supernatant was diluted with water and directly injected into the LC/MS/MS system. The separations were performed on a column packed with octadecylsilica (5 μm, 2.0 × 50 mm) with 0.1% formic acid and 0.1% formic acid in acetonitrile as mobile phase and the detection was performed using tandem mass spectrometry by multiple‐reaction monitoring via an electrospray ionization source. The standard curve was linear (r = 0.9982) over the concentration range 0.002–1 μg/mL. The intra‐ and inter‐assay precisions were 1.7 and 8.6%, respectively. The accuracy range was from 90.3 to 101.8%. The lower limit of quantification was 2.0 ng/mL using 50 μL of rat plasma sample. The developed analytical method was successfully applied to the pharmacokinetic study of lurasidone in rats. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of memantine in rat plasma by HPLC-fluorescence method and its application to study of the pharmacokinetic interaction between memantine and methazolamide

A sensitive high-performance liquid chromatographic method with fluorescence detection was developed to determine memantine (MT) in rat plasma. The method consists of pre-column labeling of MT with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) and a clean-up step with solid-phase extraction. A good separation of DIB-MT was achieved within 12 min on an octadecylsilica (ODS) column (150 × 4.6 mm i.d.; 5 μm) with a mobile phase of acetonitrile-water (70:30, v/v). The calibration curve prepared with fluoxetine as an internal standard showed good linearity in the range of 10-400 ng/mL (r = .999). The limits of detection and quantitation at signal-to-noise ratios of 3 and 10 were 2.0 and 6.6 ng/mL, respectively. The method was shown to be reliable with precisions of <5% for intra-day and <9% for inter-day as relative standard deviation. The fluorescence property and reaction yield of authentic DIB-MT were also examined. The proposed method was successfully applied to study the pharmacokinetic interaction between MT and methazolamide.     

Reversed phase UHPLC/ESI-MS determination of oxylipins in human plasma: a case study of female breast cancer

The ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method was optimized and validated for the determination of oxylipins in human plasma using the targeted approach with selected reaction monitoring (SRM) in the negative-ion electrospray ionization (ESI) mode. Reversed phase UHPLC separation on an octadecylsilica column enabled the analysis of 63 oxylipins including numerous isomeric species within 12-min run time. The method was validated (calibration curve, linearity, limit of detection, limit of quantification, carry-over, precision, accuracy, recovery rate, and matrix effect) and applied to 40 human female plasma samples from breast cancer patients and age-matched healthy volunteers (control). Thirty-six oxylipins were detected in human plasma with concentrations above the limit of detection, and 21 of them were quantified with concentrations above the limit of quantitation. The concentrations determined in healthy controls are in a good agreement with previously reported data on human plasma. Quantitative data were statistically evaluated by multivariate data analysis (MDA) methods including principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA). S-plot and box plots showed that 13-HODE, 9-HODE, 13-HOTrE, 9-HOTrE, and 12-HHTrE were the most upregulated oxylipin species in plasma of breast cancer patients.

     

Purification of porcine proinsulin by high-performance liquid chromatography

A procedure has been developed for purification of porcine proinsulin by high-performance liquid chromatography from a preparation obtained as a side product during the Sephadex G-50 gel filtration of an impure porcine insulin preparation. Reversed-phase chromatography was carried out on octadecylsilica as the stationary phase with graded mixtures of acetonitrile or methanol-acetonitrile and phosphate buffer pH 2.4 as the mobile phase. The crude preparation separated into five different groups of proteins, the proinsulin-containing peak being identified by the co-eluting internal proinsulin marker. After purification by conventional procedures (separation, pooling, freeze drying, desalting, reprecipitation and drying) this peak fraction was rechromatographed by high-performance liquid chromatography (for final purification) to give a single peak protein which had identical electrophoretic mobility to that of commercial porcine proinsulin, and which converted to a protein with electrophoretic mobility similar to that of porcine insulin.     

Solvent and salting effects on sample preparation for the determination of fenofibric acid in human plasma by HPLC-DAD

The solvent and salting effects induced on the sample preparation procedure applied to plasma samples containing fenofibric acid and 4-chlorophenyl-4′-hydroxyphenyl methanone (internal standard) are evaluated. Sodium chloride addition during a deproteinization step using both methanol and phosphoric acid influences the recovery of the analytes as well as the selectivity of the process. The chromatographic method allows high sample volume injection (500 μl) with the focusing of both analytes in the stationary phase. The synthesized high porosity octadecylsilica material allows a fast elution gradient at 4 ml/min flow-rate and a complete analysis within 7 min. UV-detection is made at 295 nm and quantitation limit in the 20 ng/ml concentration level can be achieved. The method can be successfully applied for bioequivalence studies on fenofibrate, administrated as prodrug (fenofibric acid represents its main active metabolite) in pharmaceutical formulations. The main parameters used in studying the retention behavior of the internal standard and FEFA were also estimated.     

Effects of aliphatic amines on capillary electrochromatographic performance of tricyclic antidepressants on octadecylsilica

Adding aliphatic amines to the mobile phase improves peak symmetry and efficiency in capillary electrochromatography of tricyclic antidepressants on octadecylsilica. The most hydrophobic aliphatic amine studied, dimethyloctylamine (DMOA), was the most efficient. Despite the fact that the amine additives substantially reduced the electroosmotic flow, the retention of the analytes decreased indicating a strong competitive effect of the additives. DMOA gave the largest retention decrease, and simultaneously reduced the resolution, indicating that silanophilic interaction is significant to the separation. Highest efficiencies were obtained at the lowest pH (2.8). Acetonitrile influenced both efficiency and peak symmetry, and best results were obtained at 60%.     

Analysis of low molecular weight aldehydes in air samples by capillary electrophoresis after derivatization with 4-hydrazinobenzoic acid

This work reports the analysis of selected aldehydes in air samples using capillary electrophoresis (CE). The method is based on the reaction of aldehydes with 4-hydrazinobenzoic acid (HBA) to give the corresponding hydrazones with maximum absorbance at 290 nm. Under optimized CE conditions, the HBA derivatives of four carbonyls (formaldehyde, acetaldehyde, propionaldehyde, and acrolein) were completely separated from one another, in less than 6 min, using a pH 9.3 tetraborate buffer at 0.040 mol L(-1) concentration as background electrolyte. A few method validation parameters were determined revealing good migration time repeatability (< 1.5% CV) and area repeatability (< 2% CV), excellent linearity (50-300 microg/L, r > 0.996) and adequate sensitivity for environmental applications. The limits of detection with respect to each single aldehyde were in the range of 2.7-8.8 ng L(-1). The methodology was applied to the determination of aldehydes indoors. Samples were collected in HBA impregnated octadecylsilica cartridges, at different times during the day. The most abundant carbonyls in the samples were acetaldehyde followed by formaldehyde, with estimated peak concentrations of 4.3 and 2.9 ppbv, respectively.     

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.     

Determination of dextropropoxyphene and norpropoxyphene in plasma by high-performance liquid chromatography.

A simple and sensitive procedure for the routine assay of the analgesic drug dextropropoxyphene and its main metabolite, norpropoxyphene, in plasma is described. After liquid-liquid extraction from alkalinized plasma and back-extraction into a small volume of an acidic aqueous phase, the aqueous phase was injected into a column packed with 3-microns octadecylsilica particles. Ultraviolet absorbance detection at 210 nm was used. Concentrations down to 2 nM could be determined for both compounds; at this level, the intra-assay coefficient of variation was 5%.     

Measurement of adenosine, inosine and hypoxanthine in human term placenta by reversed-phase high-performance liquid chromatography

An analytical method was developed for measuring adenosine, inosine and hypoxanthine in freshly delivered human term placentas. Representative freeze-clamped samples were taken from the sub-maternal surface of each placenta. Acid-soluble extracts of the samples were analyzed by reversed-phase high-performance liquid chromatography on columns packed with 10-micron porous octadecylsilica, using gradient elution with a linear increase in methanol concentration in ammonium phosphate buffer. Resolution of hypoxanthine from xanthine and adenosine from adenine, and quantitation of hypoxanthine and adenosine were achieved using 0.05 M ammonium dihydrogen phosphate, pH 6.5, as the low-strength eluent. Resolution of inosine from a prominent peak of beta-NAD was optimized using 0.02 M ammonium dihydrogen phosphate, pH 5.6, as low-strength eluent. Recovery of standards was greater than 90%. Mean contents (+/- S.D.) of the analytes in placentas from seven normal deliveries were, adenosine 30.6 +/- 11.5 nmol/g, inosine 68.0 +/- 25.8 nmol/g and hypoxanthine 217 +/- 127.5 nmol/g.     

Optimization of micro‐HPLC peak focusing for the detection and quantification of low hepcidin concentrations

Micro‐high‐performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 μL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile‐phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.     

Automatic Determination of Indomethacin in Human Plasma Using Liquid-Solid Extraction on Disposable Cartridges in Combination with HPLC

Abstract

A sensitive and automatic method for the analysis of indomethacin in plasma has been developed using liquid-solid extraction (LSE) on disposable extraction cartridges (DECs) coupled to high-performance liquid chromatography (HPLC). The fully automated system handles the plasma samples by performing the same operations as in a manuel procedure by means of an autosampler equipped with a robotic arm at which is attached a needle dispensing the different liquids. The DEC is first conditioned with methanol and phosphate buffer pH 7.4. A 1.0-mL volume of plasma is then applied onto the DEC; the latter is washed with the same buffer before the elution with 0.25 mL of methanol. The eluting strength of the eluate is reduced by dispensing 0.30 mL of phosphate buffer pH 7.4 in the collection tube prior to the injection onto the HPLC column via a 0.1-ml loop. The chromatographic separation is performed on an octadecylsilica column with a mixture of methanol and phosphate buffer pH 7.4 as mobile phase (60:40, v/v) and indomethacin is monitored photometrically at 254 nm. The effect of the plasma dispensing flow rate on the drug recovery and the importance of the guard column for the stability of the analytical column have been studied. The absolute recovery of the drug is 96.0 9s and the limit of detection, 2 ng/mL. At the concentration of 100 ng/mL, relative standard deviations of 1.5% (with in-day) and 2.3% (between-day) have been obtained.