Determination of morphine in cerebrospinal fluid and plasma by high-performance liquid chromatography with electrochemical detection

Two methods for the extraction of morphine from cerebrospinal fluid or plasma with quantitation by high-performance liquid chromatography with electrochemical detection were compared for accuracy, precision and ease of preparation. One procedure was a standard extraction procedure and the other utilized a commercially available liquid—liquid extraction column. Both methods produced linear calibration curves over the concentration range of 1–200 ng/ml with coefficients of correlation of 0.999. Since the electrochemical detector is capable of detecting 20 pg of morphine, biological samples as small as 0.1 to 0.4 ml can be quantified with an average relative precision of 4.1 ± 3.9% over the concentration range 1–200 ng/ml. The potential clinical importance of the assay is demonstrated using a time course distribution study of morphine in the cerebrospinal fluid and plasma of a Rhesus monkey.     

反相液相色谱法比较患者头孢吡肟的血浆和脑脊液浓度

目的:为颅脑外科术后感染患者使用头孢吡肟的合理性提供理论依据.方法:反相液相色谱法测定颅脑外科术后患者脑脊液中头孢吡肟浓度,并与其血药浓度做统计学比较.结果:头孢吡肟在患者脑脊液中的浓度为血浆中的14.6%±4.37,其在患者术后脑脊液中浓度高于常见致病菌的最低有效抑菌浓度.结论:头孢吡肟适用于颅脑外科术后患者的颅内细菌感染.     

Determination of puerarin in human plasma by high performance liquid chromatography

Puerarin, an isoflavone C-glycoside, has been identified as the major active component isolated from Pueraria lobata (Kudzu) responsible for suppression of alcohol drinking. In order to conduct clinical studies of Kudzu's efficacy, a method for measuring its bioavailability and pharmacokinetic profile is needed. We have developed a gradient reversed-phase HPLC system for pharmacokinetic study of puerarin in human plasma. Solid-phase extraction was performed on an abselut Nexus cartridge (60 mg/3 ml) possessing adsorbent function with a recovery of >97% and 4-hydroxybenzoic acid was used as an internal standard. The HPLC assay was performed on a YMC ODS-A column (150 mm x 4.6mm i.d., 5 microm particle size). The HPLC mobile phase consisted of methanol/0.5% acetic acid with 20-35% methanol gradient at a flow-rate of 0.8 ml/min. The UV wavelength was set at 254 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a puerarin concentration range of 5-500 ng/ml in human plasma. The lower limit of quantification was ca. at 8 ng/ml of puerarin in plasma. The detection limit (defined as signal-to-noise ratio of about 3) was approximately 3 ng/ml. The preliminary pharmacokinetic study after oral administration of the Kudzu capsules containing 400mg of puerarin to a healthy volunteer confirmed that the present method was suitable for determining puerarin in human plasma.     

Analysis of sofalcone in human plasma by high performance liquid chromatography

A simple, rapid, sensitive and reliable high performance liquid chromatography (HPLC) method for the determination of the anti-ulcer drug sofalcone in human plasma was developed. Plasma was extracted with ethyl acetate under acidic conditions and sofalcone was determined by HPLC using a C18 column and (methanol-0.1% formic acid aqueous 80:20) mobile phase. The linear calibration curves of sofalcone in human plasma were obtained over the concentration range of 0.01-5.0 microg/ml. The lower limit of quantitation (LLOQ) was 10 ng/ml in human plasma. The precision measured for plasma was within 15%. Extraction recovery was over 85% in blood. The method was successfully applied to the identification and quantification of sofalcone in pharmacokinetic studies.     

Tetrahydrocurcumin in plasma and urine: quantitation by high performance liquid chromatography

Tetrahydrocurcumin (THC), one of the major metabolites of curcumin, exhibits many of the same physiologic and pharmacological activities as curcumin and in some systems may exert greater antioxidant activity than curcumin. However, evaluation of clinical efficacy is limited by lack of sensitive methods for quantifying intake/absorption in blood or urine. We have developed a sensitive high performance liquid chromatography (HPLC) analytical method for detection of THC in plasma and urine. The method involves extracting the THC from 0.2 mL samples with 95% ethyl acetate/5% methanol, and beta-17-estradiol acetate as an internal standard. Analysis with a reversed-phase C18 column and UV detection at 280 nm demonstrates linear performance from 0.050 to 6.0 microg/mL in plasma, and 0.060 to 6.0 microg/mL in urine. The coefficients of variation for intra- and inter-assays were each<8.6%. The average recovery of THC from plasma and urine was greater than 98.5%. These data demonstrate a rapid, sensitive and accurate method for HPLC quantification of THC in plasma and urine.     

Measurement of resiniferatoxin in cerebrospinal fluid by high-performance liquid chromatography

A sensitive and simple high-performance liquid chromatographic (HPLC) assay was developed for the quantification of resiniferatoxin (RTX) in canine cerebrospinal fluid (CSF). A reversed-phase C(18) column and acetonitrile in 0.02 M NaH(2)PO(4) as mobile phase provided satisfactory resolution for RTX analysis. Direct HPLC analysis of the CSF samples without sample extraction or preparation improves the accuracy and detection limits of this assay. This assay was applied to measure CSF RTX content to test this method for research and clinical applications related to studies examining its analgesia effects.     

Separation of opioid peptides utilizing high performance liquid chromatography.

Chromatographic procedures have been developed for resolving all of the known enkephalins and endorphins on a single column. The effect of eluant pH on the retention times and separation of the enkephalins and beta-endorphin was determined. By combining these separations with a sensitive radioreceptor assay it is possible to assay all of the opioid peptides in the pituitary gland or in various regions of the brain from individual small laboratory animals.     

N-Acetyl-aspartylglutamate (NAAG) in human cerebrospinal fluid: Determination by high performance liquid chromatography,and influence of biological variables

Summary NAAG is one of the neuropeptides found in highest concentrations in the CNS. The presence of micromolar concentrations of NAAG in human CSF was demonstrated by using two different and complementary analytical approaches: 1) isocratic separation of endogenous NAAG by reverse-phase high performance liquid chromatography (HPLC) with dual wavelength detection and 2) derivatization of endogenous NAAG with acidic methanol and subsequent HPLC analysis of the derivative NAAG-trimethyl ester. The NAAG concentration was between 0.44µmol/l and 7.16µmol/l (mean of 2.19 ± 1.53µmol/l) in CSF samples from forty neuropsychiatric patients. Endogenous NAAG or [³H]NAAG added to CSF samples were not significantly degraded when the CSF was incubated at 37°C during one hour, suggesting that the peptide is a highly stable metabolite in the subarachnoid space. In addition, evidence is provided that NAAG does not present a concentration gradient along the lower subarachnoid space.     

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.     

Purification of integral plasma membrane proteins by reverse-phase high performance liquid chromatography

Following dissolution in anhydrous trifluoroacetic acid, plasma membrane isolated from two eukaryotic species was directly injected onto a reverse-phase high performance liquid chromatograph column. Upon development with a 60 to 100% (v/v) linear gradient of ethanol containing 0.1% trifluoroacetic acid, most of the polypeptides eluted without retention. Only the lipids and very hydrophobic proteins were retained and resolved. Most noticeable among retained proteins was the Mr 100,000 catalytic polypeptide of each species' primary plasma membrane cation pump, the Na+,K+-ATPase of pig kidney and the H+-ATPase of Neurospora crassa hyphae. This simple 60-min procedure yielded nearly pure ATPase starting from crude membranes and in a completely volatile solvent, without detergent. When fungal plasma membranes were phosphorylated in vitro with [gamma-32P]ATP prior to injection, protein kinase activity was observed and this resulted in the phosphorylation of the H+-ATPase as well as of several other less-abundant hydrophobic membrane proteins. This procedure is useful as an alternative method for the rapid characterization of those membrane-associated polypeptides that contain several hydrophobic, transmembrane sequences.     

Determination of acrylamide and glycidamide in rat plasma by reversed-phase high performance liquid chromatography

Acrylamide is a widely used monomer that produces peripheral neuropathy. It is metabolized to the epoxide, glycidamide, which is also considered to be neurotoxic. A new reversed-phase high-performance liquid chromatography (HPLC) method is described that permits simultaneous determination of acrylamide and glycidamide in rat plasma. Samples were deproteinized with acetonitrile and chromatography was performed using isocratic elution and UV absorption detection. The limits of detection for acrylamide and glycidamide were 0.05 and 0.25 μg/ml in plasma, respectively, and recovery of both analytes was greater than 90%. The assay was linear from 0.1 to 100 μg/ml for acrylamide and from 0.5 to 100 μg/ml for glycidamide. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration–time profiles of acrylamide and glycidamide in the plasma of acrylamide-treated rats.     

Characterization of cytidylyltransferase enzyme activity through high performance liquid chromatography

The cytidylyltransferases are a family of enzymes that utilize cytidine 5′-triphosphate (CTP) to synthesize molecules that are typically precursors to membrane phospholipids. The most extensively studied cytidylyltransferase is CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes conversion of phosphocholine and CTP to cytidine diphosphocholine (CDP-choline), a step critical for synthesis of the membrane phospholipid phosphatidylcholine (PC). The current method used to determine catalytic activity of CCT measures production of radiolabeled CDP-choline from ¹⁴C-labeled phosphocholine. The goal of this research was to develop a CCT enzyme assay that employed separation of non-radioactive CDP-choline from CTP. A C18 reverse phase column with a mobile phase of 0.1 M ammonium bicarbonate (98%) and acetonitrile (2%) (pH 7.4) resulted in separation of solutions of the substrate CTP from the product CDP-choline. A previously characterized truncated version of rat CCTα (denoted CCTα236) was used to test the HPLC enzyme assay by measuring CDP-choline product formation. The V_max for CCTα236 was 3850 nmol/min/mg and K_0.5 values for CTP and phosphocholine were 4.07 mM and 2.49 mM, respectively. The HPLC method was applied to glycerol 3-phosphate cytidylyltransferase (GCT) and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively.     

Determination of firocoxib in equine plasma using high performance liquid chromatography

A new method of analysis has been developed and validated for the determination of firocoxib, a new nonsteroidal anti-inflammatory drug (NSAID) approved for use in horses and dogs to control pain and inflammation associated with osteoarthritis. Following a liquid extraction using ethyl acetate:hexane (40:60), samples were separated by isocratic reversed-phase HPLC on a Sunfire C(18) column and quantified using UV detection at 290 nm. The mobile phase was a mixture of water with 0.025% trifluoroacetic acid and acetonitrile, with a flow-rate of 1.1 ml/min. The procedure produced a linear curve over the concentration range 5-1500 ng/ml with a lower limit of quantification of 5 ng/ml. Intra- and inter-assay variability was less than 7%. The average recovery was 98%. The method is suitable for the analysis of clinical samples from pharmacokinetic studies and can also be used for small volume sample sizes.     

Determination of indinavir in human cerebrospinal fluid and plasma by solid-phase extraction and high-performance liquid chromatography with column switching

A rapid, sensitive and robust sample preparation procedure for the quantitative determination of indinavir in human cerebrospinal fluid (CSF) and plasma is described. Indinavir and the internal standard were isolated from CSF or plasma samples by cation-exchange solid-phase extraction with SCX cartridges, while the chromatographic separation was adopted from a previous method, using a cyano column connected by a switching valve to a C₁₈ column. UV detection was set at 210 nm. The standard curve was linear over the concentration range of 2 to 2000 ng/ml in CSF and 5 to 2000 ng/ml in plasma. The intra-day coefficients of variation at all concentration levels were ≤5.9%. The inter-day consistency was assessed by running QC samples during each daily run. The coefficients of variation for quality control samples in both matrixes were ≤6.1%. The method has been utilized to support clinical pharmacokinetic studies.     

Simple determination of terbutaline in dog plasma by column-switching liquid chromatography

Terbutaline is a beta-adrenergic receptor antagonist that acts as a bronchodilator in the treatment of asthma and chronic bronchitis. In the present work, a column-switching high-performance liquid chromatographic method was developed to monitor terbutaline sulphate in dog plasma. The system consists of a C2 pre-column (PC) and a C18 analytical column connected in series via a switching valve. Atenolol was used as the internal standard. Good linearity was achieved in the range of 5-800 ng/ml plasma. The mean intra- and inter-assay variation coefficients for this analysis were 2.3 and 4.7%, respectively. The average recovery for terbutaline was 87.4% from plasma. The mean concentration after three freeze-thaw cycles was 99.4% of the normal value. The analytical sensitivity and accuracy of this assay is adequate for characterisation of the pharmacokinetics of oral administration of terbutaline to dogs and has been successfully used to provide pharmacokinetic data using pulsatile and immediate-release tablets.     

Measurement of branched chain amino acids in blood plasma by high performance liquid chromatography

A simple and rapid high performance liquid chromatographic technique is described for the separation and quantitation of plasma branched chain amino acids. After addition of a norleucine internal standard, plasma samples are acidified with acetic acid, and amino acids are separated from proteins and other plasma components by passage of the acidified plasma through an ion exchange resin. The ammonium hydroxide eluate from the resin is dried, phenylisothiocyanate derivatives are prepared, and the amino acids are separated on a Waters reverse-phase "Pico-Tag" column with an ultraviolet detector set at 254 nm. In addition to the branched chain amino acids (leucine, valine, and isoleucine), aspartate, glutamate, serine, threonine, alanine, and methionine are quantitated with high precision and accuracy, as verified by quantitative recovery and comparison with an automatic amino acid analyzer. The advantages of the method are its simplicity, speed, stability of derivatives, high reproducibility, low per-sample cost, and the use of a simple fixed-wavelength ultraviolet detector.     

Quantitative determination of individual teicoplanin components in human plasma and cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

We have developed a simple, rapid and highly sensitive method for determining a plasma or cerebrospinal fluid (CSF) concentrations of individual teicoplanin components using reversed-phase high-performance liquid chromatography followed by electrochemical detection. A linear relationship was observed between concentrations and peak heights for the teicoplanin concentration range of 0.025-10microg/mL. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection for the major component of teicoplanin was 1.0ng/mL (signal/noise ratio >3). Daily fluctuations of standard curves (n=5) were small, with coefficients of variation of 3.3%. The intra-assay precision was 5.9% (n=5). Inter-assay precision ranged from 2.6 to 6.8%. The method described here is suitable for clinical monitoring of teicoplanin levels in plasma or CSF level and for use in studies involving pharmacokinetics of individual teicoplanin component.     

Determination of deracoxib in feline plasma samples using high performance liquid chromatography

A new HPLC procedure for the determination of deracoxib, a selective cyclooxygenase-2 inhibitor, has been developed and validated. Following a liquid-liquid extraction using isopropyl alcohol and chloroform, samples were separated by isocratic reversed-phase HPLC on an Atlantis C18 column and quantified using UV detection at 252 nm. The mobile phase was a mixture of 10 mM potassium phosphate (pH 4.5) and acetonitrile, with a flow-rate of 1.0 ml/min. The procedure produced a linear curve over the concentration range 10-1500 ng/ml. The development of the assay allowed the determination of pharmacokinetic parameters after oral administration of deracoxib in cats and would be suitable for other pharmacokinetic studies.     

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

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