Determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and UV detection

A high-performance liquid chromatographic method with automated column switching was developed for the simultaneous determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their metabolites 13-cis-3-hydroxy-4-oxo-retinoic acid and all-trans-3-hydroxy-4-oxo-retinoic acid in plasma samples from man, rat, dog, rabbit and mouse. The method consists of deproteination of plasma (0.4 ml) with ethanol (1.5 ml), containing the internal standard Ro 12-7310. After centrifugation, 1.4 ml of the supernatant was directly injected onto the precolumn (PC) (4 x 4 mm) packed with LiChrospher 100 RP-18 (5 microm). Ammonium acetate (0.02%)-acetic acid-ethanol (100:3:4, v/v/v) was used as mobile phase M1A during injection, as well as to decrease the elution strength of the injection solution by on-line addition using a T-piece (M1B). After valve switching, the retained components were transferred to the analytical column (AC), separated by gradient elution and detected at 360 nm. Two coupled Purospher 100 RP-18 endcapped columns (both 250 x 4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid, (A), 540:450:2:30 (v/v/v/v), (B), 600:350:2:30 (v/v/v/v), and (C), 950:40:2:30 (v/v/v/v). The method was linear in the range 1-500 ng ml(-1), at least, with a quantification limit of 1 ng ml(-1). The mean recoveries from human plasma were 100-107% and the mean inter-assay precision was 2.0-4.7% (range 1-500 ng ml(-1)). Similar results were obtained for animal plasma. The analytes were stable in the plasma of all investigated species stored at -20 degrees C for 3 months, at least. The method was successfully applied to clinical and toxicokinetic studies.     

Simultaneous analysis of lignocaine and bupivacaine enantiomers in plasma by high-performance liquid chromatography

A sensitive analytical procedure is described for the simultaneous determination of lignocaine and the enantiomers of bupivacaine in biological fluids using diazepam as an internal standard. After solvent extraction into hexane, the local anaesthetics were separated using an alpha1-acid glycoprotein (AGP) column and detected at 214 nm. Calibration curves were linear (r2>0.99) in the concentration range of 5 to 500 ng/ml for the enantiomers of bupivacaine and 12.5 to 1000 ng/ml for lignocaine. The corresponding limits of detection were 4 ng/ml and 10 ng/ml, respectively. The method was applied to the analysis of plasma from a healthy woman undergoing tubal ligation.     

Simultaneous determination of p-aminohippuric acid, acetyl-p-aminohippuric acid and iothalamate in human plasma and urine by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic assay was developed for the simultaneous determination of p-aminohippuric acid (PAH), acetyl-p-aminohippuric acid (aPAH), and iothalamate in human plasma and urine. Plasma samples were prepared by protein precipitation with acetonitrile followed by evaporation, reconstitution in mobile phase, and injection onto a C18 reversed-phase column. Urine samples were diluted with 3 volumes of mobile phase prior to injection. Column effluent was monitored by UV detection at 254 nm. The lower limits of quantification in plasma were 0.5 mg/l for PAH and aPAH, and 1.0 mg/l for iothalamate. The within-day and between-day coefficients of variation in plasma and urine were < or =7.8% for all analytes. This method is well suited for renal function studies using iothalamate and PAH, whether administered as a bolus dose or by continuous infusion, to measure glomerular filtration rate and effective renal plasma flow, respectively.     

Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatography procedure

We describe a reversed-phase HPLC method for determination of total mycophenolic acid (MPA), its free concentration (MPAf), and the glucuronide metabolite (MPAG), based on simple sample preparation and gradient elution chromatography. The compounds were quantified in parallel by absorbance at 254 nm and 215 nm in the internal standard mode. Linearity was verified up to 50 mg/L for MPA and up to 500 mg/L for MPAG (r >0.999). Detection limits at 215 and 254 nm were, respectively, 0.01 and 0.03 mg/L for MPA, and 0.03 and 0.1 mg/L for MPAG. The recovery of MPA was 95-106%; recovery of MPAG was 96-106%. The imprecision (CV) for MPA (0.2-25 mg/L) was <8.4% (254 nm) and <4.4% (215 nm) within day (n = 12) and <9.2% (254 nm) and <6.2% (215 nm) between days (n = 12). The imprecision for MPAG (10-250 mg/L) was <4.9% (254 nm) and <3.4% (215 nm) within day, and <6.1% (254 nm) and <5.9% (215 nm) between days. For quantification of MPAf, 100 microL of ultrafiltrate was applied directly to the column. The detection limit was 0.005 mg/L at 215 nm and 0.015 mg/L at 254 nm. In the range between 18-210 microg/L, the within-day CVs were <11.8% (n = 12) and the between-day CVs were <15.8% (n = 12).     

Determination of amphotericin B in cerebrospinal fluid by solid-phase extraction and liquid chromatography

A highly sensitive and reproducible liquid chromatography (LC) method for the determination of Amphotericin B in cerebrospinal fluid has been developed and validated. This LC-based method involves using nystatin as an internal standard and solid-phase extraction for sample preparation, followed by reversed-phase separation monitored by absorbance at 410 nm. The method has a limit of quantification of less than 1 ng ml-1 and excellent precision and accuracy, with both percentage relative standard deviation and percentage relative error less than 10%. The established linearity range was 1-10 ng ml-1 (r2 > 0.99). The extraction recovery of Amphotericin B from the cerebrospinal fluid is higher than 90% over the entire linear range. The method has been successfully employed for studying the penetration of Amphotericin B into the central nervous system in dogs and human.     

Simultaneous quantitation of plasma doxorubicin and prochlorperazine content by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed and tested for simultaneous extraction, elution and determination of doxorubicin and prochlorperazine content in human plasma samples. The procedure consists of extraction through a conditioned C18 solid-phase extraction cartridge, elution from a Spherisorb C8 reversed-phase column by an isocratic mobile phase (60% acetonitrile, 15% methanol and 25% buffer) followed by detection with electrochemical and fluorescence detectors. Recovery of doxorubicin and prochlorperazine from pooled human plasma samples (n=3) containing 100 ng/ml of the two drugs was 77.8+/-3.5% and 89.1+/-6.0%, respectively. The lower limits of quantitation for doxorubicin and prochlorperazine in plasma samples were 6.25 ng/ml and 10 ng/ml, respectively. A linear calibration curve was obtained for up to 2 microg/ml of doxorubicin and prochlorperazine. This combination method may be of particular value in clinical studies where phenothiazines such as prochlorperazine are used to enhance retention of doxorubicin in drug resistant tumor cells.     

Simultaneous determination of guanine, uric acid, hypoxanthine and xanthine in human plasma by reversed-phase high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method with amperometric detection is described for the separation and quantification of uric acid, guanine, hypoxanthine and xanthine. The isocratic separation of a standard mixture of the compounds was achieved in 5 min on a Spherisorb 5 C18 reversed-phase column, with a mobile phase of NaH2PO4 (300 mmol dm-3, pH 3.0)-methanol-acetonitrile-tetrahydrofuran (97.8 + 0.5 + 1.5 + 0.2). Uric acid, guanine, hypoxanthine and xanthine were completely separated, with detection limits in the range 2-20 pmol per injection. The effect of pH and the composition of the mobile phase on the separation are described. The hydrodynamic voltammograms of these compounds were recorded at a glassy carbon electrode. The linear range of the calibration graph for each compound was: uric acid, 1-5000 mumol dm-3; guanine, 0.5-2000 mumol dm-3; hypoxanthine, 0.1-500 mumol dm-3 and xanthine, 0.5-5000 mumol dm-3. The within- and between-day precision was good. The uric acid and hypoxanthine content in human plasma was measured using the proposed method. Good recoveries of uric acid (97.9-103%), hypoxanthine (98.0-99.2%), guanine (96.0-98.3%) and xanthine (96.0-102%) were obtained from human plasma. The results of electrochemical detection were in good agreement with those of UV detection.     

Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

Sialosyl-fucosyl poly-LacNAc without sialosyl-Lex epitope in myeloid cell line HL60 was shown to be the ligand for E-selectin-dependent adhesion, particularly under dynamic flow conditions, in our previous study (Handa K, Stroud MR, Hakomori S, Biochemistry 36, 12412-12420, 1997). HL60 cells express only fucosyl-transferase (FT) IV and VII. X3NeuAcVII3FucnLc10, a representative component showing E-selectin-dependent binding under dynamic flow conditions, is not alpha 1-->3 fucosylated at the penultimate GlcNAc catalyzed by FT-VII, but is alpha 1-->3 fucosylated at the internal GlcNAc catalyzed by FT-IV. VI3NeuAcnLc6 is converted to VI3NeuAcIII3FucnLc6 by FT-IV, but is also converted to VI3NeuAcV3FucnLc6 by FT-VII. Thus, penultimate fucosylation catalyzed by FT-VII is not restricted for nLc6 backbone, but is highly restricted for nLc10 backbone. The cooperative effect of FT-IV and FT-VII for synthesis of poly-LacNAc having sialosyl-Lex with internal fucosylation may be blocked or highly restricted in poly-LacNAc having more than two LacNAc units, because preferential alpha 1-->3 fucosylation by FT-IV takes place at internal GlcNAc, inhibiting penultimate fucosylation by FT-VII.     

Simultaneous analysis of several non-steroidal anti-inflammatory drugs in human urine by high-performance liquid chromatography with normal solid-phase extraction

A practical and reproducible high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the simultaneous analysis of twelve non-steroidal anti-inflammatory drugs (NSAIDs) in human urine. A urine specimen mixed with acetate buffer pH 5.0 was purified by solid-phase extraction on a Sep-Pak Silica cartridge. The analyte was chromatographed by a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 5.0 as the mobile phase, and the effluent from the column was monitored at 230 or 320 nm. Absolute recoveries were greater than 73% for all of the twelve NSAIDs. The present method enabled simple manipulation and isocratic HPLC with UV analysis as well as high sensitivity of 0.005 microg/ml for naproxen, and 0.05 microg/ml for sulindac, piroxicam, loxoprofen, ketoprofen, felbinac, fenbufen, flurbiprofen, diclofenac, ibuprofen and mefenamic acid as the quantitation limit in human urine using indomethacin as an internal standard.     

Simultaneous liquid chromatography of 5-fluorouracil, uridine, hypoxanthine, xanthine, uric acid, allopurinol, and oxipurinol in plasma

A reversed-phase "high-pressure" liquid-chromatographic method is described for simultaneous analysis for 5-fluorouracil, uridine, hypoxanthine, xanthine, uric acid, allopurinol, and oxipurinol. Separation was optimal with phosphate buffer (50 mmol/L, pH 4.60) as eluent. A simple acid extraction procedure yielded quantitative recoveries and permitted adequate separation for interfering peaks. Compounds were identified by their retention times, absorbance ratios, co-elution with standards, and enzymatic shifts. With a computerized integrator we quantitated these compounds in widely varying concentrations with a single injection. The limit of sensitivity was 0.1 mumol/L for the compounds studied. This method was applied to determine mean values for those compounds in normal human plasma. They are (in mumol/L): uric acid 276 (SD 55), hypoxanthine 0.46 (SD 0.21), xanthine 0.40 (SD 0.27), and uridine 4.50 (SD 1.70). Erythrocytes and platelets can continue to release hypoxanthine and xanthine into plasma or serum after a blood specimen has been drawn. We believe this explains the higher values previously reported for hypoxanthine and xanthine in serum.     

Simultaneous measurement of allantoin, uric acid, xanthine and hypoxanthine in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for determining catabolism products of nucleic acids and purines, such as oxypurines (i.e. uric acid, xanthine and hypoxanthine) and allantoin in the blood plasma of ruminants was developed. The plasma was deproteinized with 10% trichloroacetic acid. The method enabled determination of oxypurines without derivatization. Allantoin was determined after conversion with 2,4-dinitrophenylhydrazine to a hydrazone (GLX-DNPH). Separation of converted allantoin, uric acid, xanthine and hypoxanthine derivatives was carried out using two reversed-phase C18 columns. The combination of pre-column derivatization and gradient elution with monitoring of the effluent at 205, 254 and 360 nm provides a simple and selective analytical tool for studying oxypurines and allantoin in plasma. The total run time of the HPLC analysis was 60 min. The recovery of the purine derivatives (i.e. oxypurines and allantoin) added to the plasma was between 95 and 106%. Purine derivatives were stable when the processed samples were stored for 7 days at -10 degrees C. The low values of the intra-assay coefficient of variations (2.5-4.6%) and the low values of the detection limits (0.187-0.004 nmol) point to the satisfactory precision and sensitivity of the method.     

Quantitation of acetazolamide in rat plasma, brain tissue and cerebrospinal fluid by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the analysis of acetazolamide (AZ) in rat blood (plasma/serum, whole blood and serum ultrafiltrate), brain tissue and cerebrospinal fluid (CSF) was described. Quantitative extraction of AZ with ethyl acetate from both buffered plasma and brain tissue homogenate (pH 8.0) was achieved. Each extract was evaporated to dryness and the residue was chromatographed on a reversed-phase column. CSF was directly analysed without extraction step. The limits of detection were 0.05 microgram ml-1 for plasma, 0.02 microgram g-1 for brain tissue and 0.004 microgram ml-1 for CSF. Calibration curves were linear over the working ranges of 0.1-100 micrograms ml-1 for plasma, 0.05-50 micrograms g-1 for brain tissue and 0.025-50 micrograms ml-1 for CSF. The reproducibility of AZ assay in the rat biologic media indicated very low relative standard deviations (RSDs). The recoveries of AZ added to plasma and brain tissue were more than 96% with an RSD of less than 5%. The present method was applied to studies of plasma concentration profiles of the drug after administration and its distribution into central nervous system.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C18 column (250 x 4.6 mm I.D.) using 10 mM phosphoric acid-acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 micrograms/ml and the detection limits were 0.06 micrograms/ml for indomethacin and 0.08 micrograms/ml for mefenamic acid, for 50-microliters plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

Simultaneous quantification of cefotaxime, desacetylcefotaxime, ofloxacine and ciprofloxacine in ocular aqueous humor and in plasma by high-performance liquid chromatography

Cefotaxime, given intravenously, is currently used as a broad-spectrum antibiotic for prophylaxis of intra- and postoperative infections in ocular lens surgery. A proposed therapeutic and economic alternative is the use of orally active fluoroquinolone ofloxacine as prophylactic agent. A HPLC method was developed for determination of both antibiotics in ocular aqueous humor and plasma in order to optimize dosage for safe surpassing minimal inhibitory concentration in the humor compartment. For plasma determinations a solid-phase extraction procedure was used with ciprofloxacine as internal standard. Detection limits for direct HPLC-analysis of ocular aqueous humor was 0.08 microg/ml for all compounds, whereas in plasma 0.31 microg/ml could be determined after solid-phase extraction.     

Determination of ethambutol in plasma by high-performance liquid chromatography after pre-column derivatization

A new HPLC assay using UV detection (200 nm) was developed to determine ethambutol (EMB) concentrations in plasma. Following extraction (0.1 ml plasma) with chloroform, EMB and octylamine (used as internal standard) were derivatized with phenylethylisocyanate. Quantitation in plasma was achieved at 200 nm. There were no interferences from endogenous compounds. Intra- and inter-day variabilities were lower than 5.2 and 7.6%, respectively. The limit of quantitation of the method was 0.2 microg/ml. In plasma, ethambutol was found to be stable for at least one month when samples were stored at -20 degrees C. This assay was applied to the therapeutic monitoring of EMB concentrations in 19 patients suffering from tuberculosis.     

Simultaneous analysis of hydrocortisone and hydrocortisone phosphate by high-pressure liquid chromatography: reversed-phase, ion-pairing approach

The reversed-phase, ion-pairing approach to high-pressure liquid chromatography was applied to the simultaneous analysis of hydrocortisone and its phosphate ester in laboratory-prepared samples and injectable solutions. Results of this technique were evaluated and compared with results of the official procedure.     

Assay of bropirimine in rat plasma by means of robotic solid-phase extraction and high-performance liquid chromatography

Vacuous jaw movements induced by the muscarinic agonist pilocarpine and striatal dopamine depletions were examined using a slow motion videotape system. With this procedure, rats were videotaped in a Plexiglas tube so that the profile of the head region could be seen. Vacuous jaw movements were analyzed by examining the tape at 1/6 normal speed. An observer recorded each jaw movement using a computer, and the computer program re-calculated the temporal characteristics of jaw movement responses back to normal speed. The interresponse time was recorded for each jaw movement, and each jaw movement interresponse time was assigned to a 50 ms wide time bin. Thus, the distribution of interresponse times could be used to analyze the temporal characteristics of jaw movement responses. In the first experiment, rats were administered saline vehicle, 1.0 mg/kg and 2.0 mg/kg pilocarpine. The rats were videotaped 10-15 min after injection, and the data were analyzed as described above. Pilocarpine induced very high levels of vacuous jaw movements, and the vast majority of all movements occurred in "bursts" with interresponse times of 1.0 s or less. Analysis of the interresponse time distributions showed that most of the jaw movements were within the 150-350 ms range. The modal jaw movement interresponse time was in the 150-200 ms range, which corresponds to a local frequency of 5-6.66 Hz. In the second experiment, the neurotoxic agent 6-hydroxydopamine was injected directly into the ventrolateral striatum in order to produce a local dopamine depletion. The dopamine-depleted rats were observed for jaw movements 7 days after surgery. The overall level of jaw movement activity resulting from dopamine-depletion was much lower than that produced by pilocarpine. There was a significant inverse correlation between ventrolateral striatal dopamine levels and total number of vacuous jaw movements. Videotape analysis indicated that the temporal characteristics of jaw movements induced by dopamine depletions were similar to those shown with pilocarpine. These experiments indicate that vacuous jaw movements induced by pilocarpine and striatal dopamine depletion occur in a frequency range similar to that shown in parkinsonian tremor.     

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

Age-related changes in the facial expression of pain during the first 18 months of life have important implications for our understanding of pain and pain assessment. We examined facial reactions video recorded during routine immunization injections in 75 infants stratified into 2-, 4-, 6-, 12-, and 18-month age groups. Two facial coding systems differing in the amount of detail extracted were applied to the records. In addition, parents completed a brief questionnaire that assessed child temperament and provided background information. Parents' efforts to soothe the children also were described. While there were consistencies in facial displays over the age groups, there also were differences on both measures of facial activity, indicating systematic variation in the nature and severity of distress. The least pain was expressed by the 4-month age group. Temperament was not related to the degree of pain expressed. Systematic variations in parental soothing behaviour indicated accommodation to the age of the child. Reasons for the differing patterns of facial activity are examined, with attention paid to the development of inhibitory mechanisms and the role of negative emotions such as anger and anxiety.