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.     

Simple and sensitive liquid chromatography-tandem mass spectrometry method for determination of the S(+)- and R(-)-enantiomers of baclofen in human plasma and cerebrospinal fluid

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to determine the enantiomers of the muscle relaxant baclofen in human plasma and cerebrospinal fluid (CSF) has been developed. A commercially available ultrafiltration membrane is used to prepare the sample. A chiral CROWNPAK CR(+) stationary phase column is then used to perform complete resolution of the S(+)- and R(-)-enantiomers of baclofen. This method was used to analyze human plasma and CSF spiked with baclofen, and the calibration curves for both biologic samples were linear over a concentration range of 0.15-150 ng enantiomer/ml. The lower limit of quantification was 0.15 ng enantiomer/ml in both fluids. Finally, the method was tested with an artificial CSF as an alternative to authentic human CSF. The results showed that no matrix effects and no interfering peaks were observed using this artificial CSF.     

High-performance liquid chromatographic–tandem mass spectrometric method for the determination of ethionamide in human plasma, bronchoalveolar lavage fluid and alveolar cells

We have developed and validated an accurate, sensitive, and rapid high-performance liquid chromatographic–tandem mass spectrometric method (HPLC–MS–MS) for the determination of ethionamide in plasma, bronchoalveolar fluid (BAL) and alveolar cells (AC). The retention times for ethionamide, clemastine fumarate (internal standard for plasma), promethazine (internal standard for plasma) and propranolol (internal standard for BAL and AC) were approximately 2.62, 1.21, 2.14, and 2.22 min, respectively, with a total run time of 3.2 min. Ethionamide detection for plasma was carried out on a PE Sciex API III (Perkin-Elmer, Foster City, CA, USA). BAL and cell pellets and some plasma specimens were analyzed on a Micromass Quattro LC (Micromass Co., Manchester, UK). The detection limits for ethionamide were 0.05 μg/ml for plasma, and 0.005 μg/ml for BAL supernatants and alveolar cell suspensions.     

A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid

24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker.     

A capillary liquid chromatographic/tandem mass spectrometric method for the quantification of gamma-aminobutyric acid in human plasma and cerebrospinal fluid

A sensitive and reliable method for the determination of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in human plasma and cerebrospinal fluid (CSF) has been developed. The method is based on capillary liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using deuterium-labeled GABA (gamma-aminobutyric acid-2,2-D(2), GABA-d(2)) as internal standard. Pre-column derivatization with 7-fluoro-4-nitrobenzoxadiazole (NBD-F) was deployed, allowing both effective in-line pre-concentration and sensitive tandem MS detection of the analyte. An extraction column (10 mm x 0.25 mm, 7 microm, C(18)) was used for preconcentrating and stacking the sample. Separation was carried out on an analytical column (50 mm x 0.25 mm, 5 microm, C(18)). Characteristic precursor-to-product ion transitions, m/z 267--> 249 (for NBD-GABA) and m/z 269--> 251 (for NBD-GABA-d(2)) were monitored for the quantification. A linear calibration curve from 10 to 250 ng/mL GABA with an r(2) value of 0.9994 was obtained. Detection limit was estimated to be 5.00 ng/mL GABA (S/N = 3). Human plasma and CSF samples were analyzed. The concentrations of GABA were found to be 98.6 +/- 33.9 ng/mL (mean +/- S.D., n = 12), and 44.3 +/- 10.0 ng/mL (n = 6) in plasma and CSF, respectively.     

A modified HPLC technique for simultaneous measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cerebrospinal fluid, platelet and plasma.

A sensitive, reliable and simplified HPLC assay for simultaneous measurement of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in human cerebrospinal fluid (CSF), platelets and plasma is described. Perchloric acid is used for one step precipitation of proteins and extraction of 5-HT and 5-HIAA. Precision of the assay has been increased by calibration of the instrument using serotonin-free plasma spiked with known amount of standards and N-w-methyl-5-hydroxytryptamine as internal standard. Integration of the peaks and calculations are achieved by a preprogrammed data module using ratio method. As little as 20 pg/ml of serotonin in the deproteinated sample can be detected using this procedure. In a group of surgical patients, plasma 5-HT concentration is (Mean +/- S D) 3.4 +/- 2.7 ng/ml and that of platelet 748.3 +/- 448.3 ng/10(9) platelets. In CSF, 5-HT is found to be 3.3 +/- 3.4 ng/ml and 5-HIAA is 15.1 +/- 7.3 ng/ml. A good correlation (r = 0.648, p less than .0001) is observed between 5-HT and 5-HIAA in CSF.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.     

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.     

Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry

A rapid, sensitive, and specific method using liquid chromatography tandem mass spectrometry (LC/MS/MS) has been developed for simultaneous determination of testosterone (T), dihydrotestosterone (DHT), estradiol (E2), and 5alpha-androstan-3alpha, -17beta-diol (3alpha-Diol) within human testicular fluid. Sample pretreatment involved a one-step extraction with diethyl ether. The analytes were separated on a Waters X-Terra C18 (150 mm x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (70:30, v/v) containing 0.1% formic acid using isocratic flow at 0.15 ml/min for 8 min. The column effluent was monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.1-50 ng/ml for T, 0.02-1 ng/ml for DHT, 0.05-2 ng/ml for E2, and 0.2-10 ng/ml for 3alpha-Diol, with values for the coefficient of determination of >0.99. The overall extraction efficiency was greater than 86% for T, 75% for DHT, 66% for E2, and 60% for 3alpha-Diol. The values for within-day and between-day precision and accuracy were <15%. We measured each of the four steroids in testicular sample volumes of only 20 microl, obtained by percutaneous testicular aspiration. The mean intratesticular testosterone concentration found by LC/MS/MS, 572 +/- 102 ng/ml, was similar to that previously obtained by radioimmunoassay (RIA). The mean intratesticular estradiol concentration was 15.7 +/- 2.3 ng/ml, which also correlated well with RIA measurement. Both DHT and 3alpha-Diol were below the limits of detection by RIA, but could be measured accurately by LC/MS/MS. In conclusion, LC/MS/MS represents a sensitive and accurate means by which to measure four separate steroids within small volume samples of testicular fluid.     

Effect of hCG injection on prostaglandin E concentrations in ram seminal plasma

Ram and bull seminal plasma, respectively, contain 0.5-20 microg PGE/ml and 5-10 ng PGE/ml. To demonstrate that PGE concentrations in the seminal plasma are related to sperm quality and could be affected by hormonal stimulation in vivo, four rams were injected with 500 IU hCG, in and out of season. The rams responded 1 week after hCG with a 1.5- to 4-fold increase in seminal plasma PGE. The PGE peak was temporally separate from the hCG-induced rise in seminal plasma testosterone which was observed after 1 day. Using a simulated cryptochid ram, peaks in seminal fluid PGE were found to be associated with increased sperm velocity and sperm counts. In bulls, PGE concentrations in the seminal plasma of good bulls were significantly higher than that found in poor and cryptorchid bulls.     

Disassociated increases of adrenomedullin in the rat cerebrospinal fluid and plasma after salt loading and systemic administration of lipopolysaccharide

To determine the role of adrenomedullin (AM) in the fluid electrolyte homeostasis and endotoxin shock, cerebral spinal fluid (CSF) and plasma were sampled from rats after respective challenges. The AM levels were measured by a highly sensitive immunoassay. The AM levels in the CSF of the rats anesthetized with ether (10.7 +/- 0.60 fmol/ml) were significantly higher than those with isoflurane 5.17 +/- 0.70 fmol/ml, P < 0.01), while the plasma level did not differ significantly. The CSF levels of the rats received 2% saline drinking increased to 3 and 4 folds at day 5 and day 7, respectively, while the plasma levels did not differ from controls at both time points. The AM levels in CSF or plasma increased to 1.5 and 3 folds at 1.5 h after intraperitoneal (i.p.) administration of lipopolysaccharide (LPS, 5 mg/kg), reached 6.5 and 30 folds at 6 h, respectively, while no change was observed in the controls. The present findings suggest that AM in the CSF is regulated independently from that in the plasma, the centrally synthesized AM plays and important role in the regulation of the fluid electrolyte homeostasis. Furthermore, the circulatory AM plays an important role in the endotoxin shock.     

Detection of prostatic tissue and seminal plasma peptides reacting with human seminal fluid acid phosphatase antibodies

IgG1 monoclonal antibody to purified seminal fluid phosphatase was raised by fusion of spleen cells from immunized mice with cell line Sp2/O-Ag 14 using simple method of screening for antiphosphatase antibody secreting clones. All molecular forms of catalytically active seminal fluid phosphatase and prostatic tissue phosphatase, resolved by chromatofocusing in pH gradient, react with this monoclonal antibody and with rabbit antiserum to purified seminal fluid phosphatase. Peptides of Mr 25,000 to 76,000 and of Mr 13,000 to 76,000 were adsorbed from the prostatic tissue extract and from seminal plasma on the monoclonal antibody-Sepharose column.     

Determination of vandetanib in human plasma and cerebrospinal fluid by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive and precise LC-ESI-MS/MS method for the determination of vandetanib (ZD6474) in human plasma and cerebrospinal fluid (CSF) using [(13)C,d(3)]-ZD6474 as an internal standard (ISTD) was developed and validated. Sample preparation consisted of a simple liquid-liquid extraction with tert-butyl methyl ether containing 0.1% or 0.5% ammonium hydroxide. ZD6474 and ISTD were separated on a Kinetex C18 column (2.6 μm, 50 mm × 2.1 mm) at ambient temperature with an isocratic mobile phase (acetonitrile/10mM ammonium formate=50/50, v/v, at pH 5.0) delivered at 0.11 mL/min. The retention time of both compounds was at 1.60 min in a runtime of three min. Detection was achieved by an API-3200 LC-MS/MS system, monitoring m/z 475.1/112.1 and m/z 479.1/116.2 for vandetanib and ISTD, respectively. The method was linear in the range of 0.25-50 ng/mL (R(2) ≥ 0.990) for the CSF curve and from 1.0 to 3000 ng/mL (R(2) ≥ 0.992) for the plasma curve. The mean recovery for vandetanib was 80%. Within-day and between-day precisions were ≤ 8.8% and ≤ 5.9% for CSF and plasma, respectively. Within-day and between-day accuracies ranged from 95.0 to 98.5% for CSF, and from 104.0 to 108.5% for plasma. Analysis of plasma from six different sources showed no matrix effect for vandetanib (MF=0.98, %CV ≤ 4.97, n=6). This method was successfully applied to the analysis of pharmacokinetic samples from children with brain tumors treated with oral vandetanib.     

Effects of pregnancy and labor on oxytocin levels in human plasma and cerebrospinal fluid

In order to investigate the significance of oxytocin in pregnancy and labor, oxytocin concentrations in plasma and cerebrospinal fluid (CSF) were determined using the specific radioimmunoassay. Plasma and CSF samples were obtained from 23 pregnant women (11 pre labor, 12 in labor), 15 nonpregnant women and 4 men at spinal puncture for anesthesia. In males and nongravidas, CSF levels of oxytocin were significantly higher than plasma levels. Plasma levels in pregnant patients pre or in labor were significantly higher than those in nongravidas. No significant difference between CSF levels in prelabor gravidas (mean +/- SE, 9.7 +/- 1.5 mu u/ml) and nongravidas (10.1 +/- 1.2 mu u/ml) was found. However, CSF levels in gravidas in labor (18.6 +/- 2.3 micromicrons/ml were significantly higher than the levels in prelabor gravidas. These results strongly suggest that oxytocin levels in human plasma and CSF are controlled by different mechanisms and that the increased oxytocin could have some specific central actions.     

Quantitative determination of hederagenin in rat plasma and cerebrospinal fluid by ultra fast liquid chromatography-tandem mass spectrometry method

A rapid, sensitive and selective method was developed for the quantitative determination of hederagenin in rat plasma and cerebrospinal fluid (CSF) by ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). It has been successfully applied in a pharmacokinetic study of hederagenin in the central nervous system (CNS). Sample pretreatment involved a simple protein precipitation with methanol and a one-step extraction with ethyl acetate. Separation was carried out in a Shim-pack XR-ODS II (75 mm × 2.0 mm, i.d., 2.1 μm) column with gradient elution at a flow rate of 0.35 mL/min. The mobile phase was 5mM ammonium acetate and acetonitrile. Detection was performed in a triple-quadruple tandem mass spectrometer by multiple-reaction-monitoring mode via electrospray ionization. A linear calibration curve for hederagenin was obtained over a concentration range of 0.406 (lower limit of quantification, LLOQ) to 203 ng/mL (r2 > 0.99) for both plasma and CSF. The intra-day and inter-day precision (relative standard deviation, RSD) values were less than 15%. At all quality control (QC) levels, the accuracy (relative error, RE) was within -9.0% and 11.1% for plasma and CSF, respectively. The pharmacokinetics results indicated that hederagenin could pass through the blood-brain barrier. This UFLC-MS/MS method demonstrates higher sensitivity and sample throughput than previous methods. It was also successfully applied to the pharmacokinetic study of hederagenin following oral administration of Fructus akebiae extract in rats.     

Transferrin in fetal sheep cerebrospinal fluid and plasma during gestation

1. Transferrin concentrations in fetal sheep CSF and plasma have been estimated between 31 and 125 days gestation and in the adult, using a radial immunodiffusion assay. 2. The plasma concentration was lowest (183 +/- 35 mg/100 ml) in the earliest fetuses examined (31 days). It increased to over 350 mg/100 ml by 35 days; thereafter it was around the adult value (580 mg/100 ml). 3. In CSF the transferrin concentration increased from 43 +/- 10 mg/100 ml at 31 days to a maximum of 163 +/- 14 mg/100 ml at 40 days gestation after which it decreased considerably to 6.1 +/- 0.7 mg/100 ml at 125 days and was even lower in the adult (1.1 +/- 0.2 mg/100 ml). 4. CSF: plasma ratios for transferrin especially when compared with those of other plasma proteins, are not compatible with passive leakage of protein from blood to CSF in the developing brain. The results may be explained by specific transfer of proteins into CSF but synthesis by the choroid plexus or brain has not been excluded.     

Lipid peroxidation products and antioxidant proteins in plasma and cerebrospinal fluid from multiple sclerosis patients

Lipid peroxidation (LPx) products were measured as thiobarbituric acid-reactive substances (TS) and lipid-soluble fluorescent pigments (FP) in both plasma and CSF from MS patients and controls. Although no significant changes were found in MS plasma, we report here for the first time increases in both TS and FP in MS CSF (p<0.05 and p<0.01, respectively, compared with patients with other neurological diseases), indicating that increased LPx in CNS may be a feature of MS. Levels of transferrin were normal but caeruloplasmin (CP), a major antioxidant plasma protein, was significantly raised in MS patients (p<0.01) and this may represent an adaptive response to increased oxidative challenge. Neither of these proteins was detectable in CSF using radial immunodiffusion. There was no significant correlation between the severity or duration of the disease nor the period since the last relapse and either LPx products of CP suggesting that the changes observed in this work are not simply the direct result of demyelination and tissue damage.     

Development of cerebrospinal fluid and the blood-cerebrospinal fluid barrier in rabbits.

Blood plasma and cerebrospinal fluid (CSF) samples were collected from adult female rabbits (New Zealand White), newborn, and embryos at 18, 20, 24, and 28 days of gestation. Samples were analyzed for total protein using the Folin phenol reagent. During development, mean total protein of blood plasma rose sharply from 12.45 to 12.51 mg/ml at 18 to 20 days to 37.56 mg/ml at 28 days. Levels further increased to 54.06 mg/ml in the newborn and to 66.18 mg/ml in the adult. The protein concentration of cerebrospinal fluid was constant at 5.20 to 5.29 mg/ml between 18 and 20 days of gestation, but steadily decreased to 3.53 mg/ml at 28 days. By birth, the CSF protein concentration was further reduced to 2.08 mg/ml, and this level differed only slightly (P < 0.05) from CSF protein values determined for adults. These data indicate that the blood-cerebrospinal fluid barrier to proteins begins to function by 18 to 20 days of gestation, and the protein concentration of cerebrospinal fluid approaches the normal adult value soon after birth.