LC-ESI-MS-MS Method for Monitoring Dopamine,Serotonin and Their Metabolites in Brain Tissue

A rapid and precise LC-ESI-MS-MS method for the parallel identification and quantification of dopamine, serotonin and their metabolites (homovanillic acid, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindolacetic acid) from rat brain tissue without any pre-analysis adjustment of the sample such as pre-concentration or derivatization has been developed. In particular, the reaction-monitoring mode was selected for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. Alternation the ionization polarity in the course of mass spectrometry detection enabled to determine substances susceptible to various ionization modes in only one analysis run. This fact, in combination with an easy pre-treatment step, constitutes the method straightforward and time undemanding. The developed method was characterized with a high precision (≤19.5%, determined as RSD), an acceptable accuracy (≥82.0%, determined as recovery), a low limit of detection (≤0.40 ng/100 mg brain tissue) and a low limit of quantification (≤0.42 ng/100 mg brain tissue). The method has been applied in a recent animal study. The levels of the studied neurotransmitters have been determined in the rat brain hippocampus, prefrontal cortex, and striatum in an animal model of schizophrenia induced by an acute dose of a dizocilpine.     

A GC-SIM-MS Method for the Determination of Butylidenephthalide in Rat Plasma and Tissue: Application to the Pharmacokinetic and Tissue Distribution Study

Abstract

Butylidenephthalide is one of the major active components isolated from Rhizoma Chuanxiong. This paper describes a simple, rapid, specific and sensitive method for the quantification of butylidenephthalide in rat plasma and tissue distribution using a liquid-liquid extraction procedure followed by capillary gas chromatography-selected ion monitoring mode-mass spectrometry (GC-SIM-MS) analysis. The calibration curves were linear over the concentration ranging from 0.02–10.0 µg/mL (r > 0.99) for plasma samples and 0.18–7.25 µg/g (r > 0.99) for the tissue samples. The limit of quantification (LOQ) was 1.0 ng/mL or 1.0 ng/g (ten times signal/noise ratio). Within- and between-day precisions expressed as the relative standard deviation (RSD) for the method were 2.39–2.98% and 2.97–4.26%, respectively. The methods of recovery for all samples were greater than 80% at the low, medium, and high concentrations. The method has been successfully applied to a pharmacokinetics study in rats after an oral administration of Butylidenephthalide with a dose of 20.0 mg/kg. The main pharmacokinetic parameters obtained were T _max  = (0.22 ± 0.06) h, C _max = (3 ± 1) µg/mL, AUC = (32 ± 6) h?µg/mL, and K _a  = (8.5 ± 0.8)/h. The results showed that the butylidenephthalide was easily absorbed. The concentrations of butylidenephthalide in rat kidney, lung, heart, and cerebellum were higher than those in other organs. To determine free fraction in serum, samples were filtered using ultrafiltration membranes with a molecular weight cut-off of 10,000 Da and extracted using liquid-liquid extraction. The extracts were evaporated and analyzed by GC-MS. The protein binding in rat plasma, human plasma, and human serum albumin were 83 ± 4%, 94 ± 3%, and 89 ± 3%, respectively.     

Determination of N-acetylaspartic acid concentration in the mouse brain using HPLC with fluorescence detection

The concentration of brain N-acetylaspartic acid (NAA) in mice was determined by high-performance liquid chromatography (HPLC) using fluorescence detection after pre-column derivatization with 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Six different brain parts, namely, the prefrontal cortex, olfactory bulb, nucleus accumbens, striatum, cerebellum and hippocampus, of male C57BL6/J mice, were investigated. The NAA concentration (nmol/mg protein) was highest in the olfactory bulb (58.2 ± 4.0, n = 8) and lowest in the hippocampus (42.8 ± 1.6, n = 8). The proposed HPLC method with fluorescence detection was successfully used to determine the NAA concentration in each investigated brain area.     

Determination of Amifostine and WR1065 in Rat Plasma by CE with Amperometric Detection

A method based on capillary electrophoresis with amperometric detection (CE–AD) was developed for the determination of amifostine (a cytoprotective agent, WR2721) and 2-(3-aminopropylamino)ethanethiol) (WR1065, the active metabolite of WR2721) in rat plasma. The contents of WR1065 and amifostine were determined by measuring WR1065 in deproteinized rat plasma using CE–AD before and after it was incubated at 37 °C for 4 h in acidic solution, respectively. During the incubation, amifostine was quantitatively converted to WR1065. In addition, cysteine and uric acid in rat plasma were also determined simultaneously. The detection electrode was a 500 μm diameter platinum disc electrode at a detection potential of +1.0 V (vs. saturated calomel electrode). The analytes can be well separated within 9 min in a 50-cm-long fused-silica capillary at a separation voltage of 18 kV in a 100 mM phosphate buffer (pH 7.5). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with the limits of quantification (S/N = 3) ranging from 0.60 to 1.40 μM. The method has been validated. Satisfactory within-day and between-day precisions were obtained with relative standard deviations of ≤4.9 and ≤5.1 % for WR1065 and ≤5.0 and ≤5.3 % for amifostine, respectively. The within-day and between-day accuracy was in the range of 98.6–102.3 % and 95.7–97.2 % for WR1065 and 97.5–98.6 and 95.3–97.1 % for amifostine, respectively.

     

Determination of Histamine in Rat Plasma and Tissue Extracts by Intramolecular Excimer-Forming Derivatization and LC with Fluorescence Detection

A highly sensitive and selective method for determination of histamine in rat plasma and tissue extracts by liquid chromatography with fluorescence detection is described. The method is based on precolumn derivatization of amino groups of histamine with two molecules of 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester that allows generation of intramolecular excimer fluorescence. The detection limit for histamine was 0.183 nM. This sensitivity allowed determination of histamine in 10 μL of rat plasma or in the extracts from less than 1 mg of tissue.     

Determination of Amifostine and WR1065 in Rat Plasma by CE with Amperometric Detection

A method based on capillary electrophoresis with amperometric detection (CE–AD) was developed for the determination of amifostine (a cytoprotective agent, WR2721) and 2-(3-aminopropylamino)ethanethiol) (WR1065, the active metabolite of WR2721) in rat plasma. The contents of WR1065 and amifostine were determined by measuring WR1065 in deproteinized rat plasma using CE–AD before and after it was incubated at 37 °C for 4 h in acidic solution, respectively. During the incubation, amifostine was quantitatively converted to WR1065. In addition, cysteine and uric acid in rat plasma were also determined simultaneously. The detection electrode was a 500 μm diameter platinum disc electrode at a detection potential of +1.0 V (vs. saturated calomel electrode). The analytes can be well separated within 9 min in a 50-cm-long fused-silica capillary at a separation voltage of 18 kV in a 100 mM phosphate buffer (pH 7.5). The relation between peak current and analyte concentration was linear over about 3 orders of magnitude with the limits of quantification (S/N = 3) ranging from 0.60 to 1.40 μM. The method has been validated. Satisfactory within-day and between-day precisions were obtained with relative standard deviations of ≤4.9 and ≤5.1 % for WR1065 and ≤5.0 and ≤5.3 % for amifostine, respectively. The within-day and between-day accuracy was in the range of 98.6–102.3 % and 95.7–97.2 % for WR1065 and 97.5–98.6 and 95.3–97.1 % for amifostine, respectively.     

Determination of Protamine and Insulin Using Short-End Injection Capillary Electrophoresis

A fast and inexpensive method for simultaneous determination of total protamine and insulin has been developed using capillary electrophoresis in the short-end injection setup with a bare-fused silica capillary. Optimized background electrolyte consists of 45 mM aqueous solution of phosphoric acid, pH 1.85. Separation is finished within 1 min; total analysis time including preconditioning is 4 min. The method exhibits excellent linearity within the concentration range 2.5–500 μg mL⁻¹ for both analytes. Limits of detection are 1.0 and 0.7 μg mL⁻¹ for protamine and insulin, respectively. Accuracy of the method has been successfully tested on a real sample of Neutral Protamine Hagedorn Insulin (NPH insulin) injection. The background electrolyte employed is inexpensive and experiments have shown that it does not need to be exchanged for at least 20 subsequent analyses.

     

Decreased l-tryptophan concentration in distinctive brain regions of mice treated repeatedly with phencyclidine

It has been reported that repeated phencyclidine (PCP) treatment induces schizophrenia-like behavior in mice. l-Tryptophan (Trp) concentrations in brain tissues of control (n?=?8) and PCP-treated mice (10 mg/kg/day, s.c., 14 days, n?=?10) were determined using high-performance liquid chromatography (HPLC) with fluorescence detection. The HPLC method involved pre-column fluorescence derivatization with (R)-(?)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-PyNCS). Eight different parts of the brain, namely, the frontal cortex, nucleus accumbens, striatum, hippocampus, amygdala, thalamus, hypothalamus, and cerebellum, of both groups were investigated. A significant decrease in the l-Trp concentration in the nucleus accumbens (p?=?0.024) and hippocampus (p?=?0.027) was observed in PCP-treated mice, suggesting that alteration of the l-Trp metabolism might occur in these brain parts.     

Characterization of the Interaction Between Lantibiotics, Nisin and Duramycin, and β-Lactoglobulin by Affinity Capillary Electrophoresis

Affinity capillary electrophoresis was used to study quantitatively the noncovalent interactions between β-lactoglobulin (β-LG), a milk whey protein, and two lantibiotics, nisin (a dairy biopreservative lantibiotic) and duramycin (a bovine mastitis treatment lantibiotic). The study involved measuring the change in effective electrophoretic mobility of the lantibiotic as the concentration of β-LG in the background electrolyte is increased. Nonlinear regression analysis was used to model the dependence of the effective mobility of the lantibiotic on β-LG concentration in the BGE. Using this approach, binding constants were determined to be 3.1 (±0.2) × 10⁸ M^?1 for nisin and 2.2 (±0.1) × 10⁸ M^?1 for duramycin. Both binding constants were comparable indicating the similarity of affinity properties of nisin and duramycin towards β-LG. These results demonstrate that affinity capillary electrophoresis is a suitable method for characterizing the interaction between lantibiotics and β-LG.     

LC Determination of a Novel Synthetic Thiazolidinedione (BP-1107) in Rat Plasma and Its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatographic method for the quantification of BP-1107 in rat plasma has been established. Plasma samples were prepared by extraction with tert-butyl methyl ether, and troglitazone was used as an internal standard. The analytical separation was performed on a C₁₈ column using acetonitrile–0.3% phosphoric acid in water (pH 4.00 adjusted with triethylamine) (75:25, v/v) as a mobile phase. A detailed validation of the method was performed as per USFDA guidelines. For BP-1107 at the concentrations of 2.42, 16.11 and 32.22 μg mL^?1 in rat plasma, the extraction recoveries were 114.14 ± 9.75, 95.37 ± 12.06 and 90.00 ± 6.46%, respectively. The mean recovery for internal standard was 91.96 ± 2.51%. The lower limit of quantitation of BP-1107 was 16 ng. The linear quantification range of the method was 0.81–53.70 μg mL^?1 in rat plasma with a correlation coefficient greater than 0.999. The intra-day and inter-day accuracy for BP-1107 at 2.42, 16.11 and 32.22 μg mL^?1 levels in rat plasma fell between 97.10–110.02 and 97.52–108.04%. The intra-day and inter-day precision were in the ranges of 1.91–5.63 and 4.43–6.28%, respectively. The method was successfully applied to a pharmacokinetic study of BP-1107 in rats after an intravenous administration.     

Characterization of the Interaction Between Lantibiotics,Nisin and Duramycin,and β-Lactoglobulin by Affinity Capillary Electrophoresis

Affinity capillary electrophoresis was used to study quantitatively the noncovalent interactions between β-lactoglobulin (β-LG), a milk whey protein, and two lantibiotics, nisin (a dairy biopreservative lantibiotic) and duramycin (a bovine mastitis treatment lantibiotic). The study involved measuring the change in effective electrophoretic mobility of the lantibiotic as the concentration of β-LG in the background electrolyte is increased. Nonlinear regression analysis was used to model the dependence of the effective mobility of the lantibiotic on β-LG concentration in the BGE. Using this approach, binding constants were determined to be 3.1 (±0.2) × 10⁸ M⁻¹ for nisin and 2.2 (±0.1) × 10⁸ M⁻¹ for duramycin. Both binding constants were comparable indicating the similarity of affinity properties of nisin and duramycin towards β-LG. These results demonstrate that affinity capillary electrophoresis is a suitable method for characterizing the interaction between lantibiotics and β-LG.

     

Determination of Tectorigenin in Rat Plasma: Application to a Pharmacokinetic Study After Oral Administration of Tectorigenin or Its Prodrug Tectoridin

A simple, sensitive, and validated liquid chromatographic method has been developed for the determination of tectorigenin in rat plasma and application to a pharmacokinetic study after oral administration of tectorigenin or its prodrug tectoridin. The analysis was performed on a Kromasil C₁₈ analytical column using gradient elution with acetonitrile 0.1% phosphonic acid water at 0.8 mL min^?1. The detection wavelength for UV detection was set at 264 nm. The established method was fully validated with parameters as follows: the intra- and inter-day assay precisions (CV) of three analytes were in the range of 4.2–13.3% and accuracies were between 98.0 and 107.5%; the calibration curve was linear with r ² > 0.99 over a concentration range of 0.02–2 μg mL^?1; the lower limit of quantification was 0.02 μg mL^?1; tectorigenin showed stable in rat plasma after 12 h incubation at room temperature, 15 days storage at ?80 °C and three freeze/thaw cycles, as well as in reconstitute buffer for 24 h at 25 °C; and the mean recoveries of tectorigenin were 92.3 ± 3.2, 95.5 ± 2.9 and 94.5 ± 3.0% with quality control levels of 0.02, 0.2 and 2 μg mL^?1, respectively. In conclusion, this method is simple, economic, and sensitive enough for in vivo pharmacokinetic studies of tectorigenin.     

HPLC Analysis and Pharmacokinetic Study of Paeoniflorin after Intravenous Administration of a New Frozen Dry Powder Formulation in Rats

A simple and specific high performance liquid chromatographic (HPLC) method with UV detection using picroside II as the internal standard was developed and validated to determine the concentration of paeoniflorin in rat plasma and study its pharmacokinetics after an single intravenous administration of 40 mg kg^?1 paeoniflorin to Wistar rats. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 0.05 mol L^?1 NaH₂PO₄ solution (pH 5.0). Chromatographic separation was achieved on an Agilent XDB C₁₈ column (250 × 4.6 mm I.D., 5 μm) with a Shim-pack GVP-ODS C₁₈ guard column (10 × 4.6 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–water–acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 mL min^?1. The UV detection was performed at a wavelength of 230 nm. The linear calibration curves were obtained in the concentration range of 0.05–200.0 μg mL^?1 in rat plasma with the lower limit of quantification (LLOQ) of 0.05 μg mL^?1. The intra- and inter-day precisions in terms of % relative standard deviation (RSD) were lower than 5.7 and 8.2% in rat plasma, respectively. The accuracy in terms of % relative error (RE) ranged from ?1.9 to 2.6% in rat plasma. The extraction recoveries of paeoniflorin and picroside II were calculated to be 69.7 and 56.9%, respectively. This validated method was successfully applied to the pharmacokinetic study of a new paeoniflorin frozen dry power formulation. After single intravenous administration, the main pharmacokinetic parameters t _1/2, AUC_0-∞, CL_TOT, V _Z, MRT_0-∞ and V _ss were 0.739 ± 0.232 h, 43.75 ± 6.90 μg h mL^?1, 15.50 ± 2.46 L kg^?1 h^?1, 1.003 ± 0.401 L kg^?1, 0.480 ± 0.055 h and 0.444 ± 0.060 L kg^?1, respectively.     

Development and validation of a capillary electrophoresis method for the characterization of sulfoethyl cellulose

To characterize sulfoethyl cellulose el samples, a capillary electrophoresis method was developed and validated sulfoethyl cellulose el was hydrolyzed, and the resulting d ‐glucose derivatives were analyzed after reductive amination with 4‐aminobenzoic acid using 150 mM boric acid, pH 9.5, as background electrolyte at 20°C and a voltage of 28 kV. Peak identification was derived from capillary electrophoresis with mass spectrometry using 25 mM ammonia adjusted to pH 6.2 by acetic acid as electrolyte. Besides mono‐, di‐, and trisulfoethyl d ‐glucose small amounts of disaccharides could be identified resulting from incomplete hydrolysis. The linearity of the borate buffer‐based capillary electrophoresis method was evaluated using d ‐glucose in the concentration range of 3.9–97.5 μg/mL, while limits of detection and quantification derived from the signal‐to‐noise ratio of 3 and 10 were 0.4 ± 0.1 and 1.2 ± 0.3 μg/mL, respectively. Reproducibility and intermediate precision were determined using a hydrolyzed sulfoethyl cellulose el sample and ranged between 0.2 and 8.8% for migration times and between 0.3 and 10.4% for peak area. The method was applied to the analysis of the degree of substitution of synthetic sulfoethyl cellulose el samples obtained by variation of the synthetic process and compared to data obtained by elemental analysis.     

Optimization of Capillary Isotachophoretic Method for Histidine Determination in Protein Matrices

The capillary isotachophoretic method was optimized and used for histidine determination in food samples. The optimum conditions for histidine separation and determination were found on the experimental conditions such as: selectivity, separation speed, pH, concentration of the leading and terminating electrolytes, and electroosmotic flow additives. The optimum electrolytes composition [leading electrolyte: 7 mM NH₄OH + 15 mM 2-(N-morpholino)ethanesulfonic acid + 1% hydroxyethylcellulose; pH = 6.10 and terminating electrolyte: 15 mM aminocaproic acid +5 mM acetic acid +40% methanol; pH = 5.10] and conditions of analysis were adopted for histidine determination in food samples (meat and fish products). The proposed electrolyte system was characterized by linearity (10–100 and 100–430 mg · L^?1 with R² = 0.9976 and 0.9991), accuracy (99.5% and 98%), intra-assay of the relative step height (1.40% for standard and 3.20% for food samples analysis), inter-assay of the relative step height (3.65% and 6.30%) and satisfactory quantification and detection limits. The obtained results were compared to a chromatographic method (reversed-phase (RP)-HPLC) for determination of histidine. The average concentrations of histidine in the samples assayed by both methods were statistically comparable. It should be noted that the proposed histidine determination method can be considered as a contribution to Green Analytical Chemistry.     

Determination of Metabolites in the Cerebellum of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Exposed Mice by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin elicits many harmful effects in tissues. Metabolomic changes and the associated pathway alterations caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the cerebellum, an area thought to be less affected by environmental alterations, remain unknown. Here, metabolomics was performed to identify endogenous metabolites that were associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin in the cerebellum of 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated mice using Fourier transform ion cyclotron resonance mass spectrometry. Distinct peaks were located in two mass ranges, 210 m/z–420 m/z and 450 m/z–570 m/z. In principal component space, the high-dose group was clearly separated from the control group. Six metabolites associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin dose were identified. The metabolite 1-palmitoyl lysophosphatidic acid increased with increasing doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin, indicating activation of the rat sarcoma pathway. Biosynthesis of the unsaturated fatty acid 18-hydroxyoleate was inhibited upon 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. The decrease in N-arachidonoyl taurine, implies that taurine increased, suggesting inhibition of neuronal signal transmission. A decrease in N-acetyl-aspartyl-glutamate has been associated with injury of the cerebellum through activation of N-methyl-D-aspartic acid receptors. An increase in glycerophosphoinositol suggests damage to blood–brain barrier function, and changes in purine metabolism were observed because inosine increased following 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. These results suggest that 2,3,7,8-tetrachlorodibenzo-p-dioxin activates the rat sarcoma pathway, alters fatty acid biosynthesis and purine metabolism, inhibits neurotransmitter systems, and is harmful to blood–brain barrier function in the cerebellum.     

Determination of Tectorigenin in Rat Plasma: Application to a Pharmacokinetic Study After Oral Administration of Tectorigenin or Its Prodrug Tectoridin

A simple, sensitive, and validated liquid chromatographic method has been developed for the determination of tectorigenin in rat plasma and application to a pharmacokinetic study after oral administration of tectorigenin or its prodrug tectoridin. The analysis was performed on a Kromasil C₁₈ analytical column using gradient elution with acetonitrile 0.1% phosphonic acid water at 0.8 mL min⁻¹. The detection wavelength for UV detection was set at 264 nm. The established method was fully validated with parameters as follows: the intra- and inter-day assay precisions (CV) of three analytes were in the range of 4.2–13.3% and accuracies were between 98.0 and 107.5%; the calibration curve was linear with r ² > 0.99 over a concentration range of 0.02–2 μg mL⁻¹; the lower limit of quantification was 0.02 μg mL⁻¹; tectorigenin showed stable in rat plasma after 12 h incubation at room temperature, 15 days storage at −80 °C and three freeze/thaw cycles, as well as in reconstitute buffer for 24 h at 25 °C; and the mean recoveries of tectorigenin were 92.3 ± 3.2, 95.5 ± 2.9 and 94.5 ± 3.0% with quality control levels of 0.02, 0.2 and 2 μg mL⁻¹, respectively. In conclusion, this method is simple, economic, and sensitive enough for in vivo pharmacokinetic studies of tectorigenin.

     

In Vivo Determination of Reduced Thiols in Rat Cerebellum Paraflocculus Following Salicylate-Induced Tinnitus by Fluorescence

The increasing number of patients suffering from tinnitus may be explained by an imbalance in the antioxidant defense system and reactive oxygen species formation, suggesting the importance of the redox homeostasis in this condition. Endogenous biothiols play important roles in maintaining redox homeostasis. Hence, to understand the role of biothiols in the pathological process of tinnitus, this study demonstrates an in vivo method for monitoring the concentrations of biothiols in the paraflocculus of the rat cerebellum during tinnitus induced by the injection of salicylate. Resorufin-based fluorescent probe 1 was used as the selective probe with in vivo microdialysis. Probe 1 was premixed with microdialysates from the paraflocculus of the rat cerebellum and transferred into a cuvette for continuous-flow fluorescence. A linear relationship between the fluorescence and the concentrations of cysteine, homocysteine, and glutathione was from 1 to 15?µM, with detection limits of 128, 148, and 183?nM, respectively. The basal level of total reduced biothiols in the paraflocculus of the rat cerebellum microdialysate was determined to be 10.62?±?1.34?µM based on the calibration curve for homocysteine. The injection of sodium salicylate into the animals significantly decreased the reduced biothiol concentrations in the paraflocculus of the rat cerebellum from 60?min, reaching 39.5?±?6.20% of the basal level. In contrast, at 120, 180, and 300?min, the concentrations of reduced biothiols were 55.73?±?9.35, 58.26?±?9.56, and 91.69?±?6.91% of the basal level, respectively. These results imply that the decrease in reduced biothiols in the paraflocculus of the rat cerebellum may be associated with salicylate-induced tinnitus. This study offers a new platform for in vivo monitoring of reduced biothiols in the paraflocculus of the rat cerebellum following salicylate-induced tinnitus and may be useful for the investigation of this condition.     

Simple and sensitive high-performance liquid chromatography method for the quantitation of indinavir in rat plasma and central nervous system

A simple and sensitive RP-HPLC method using UV detection (215 nm) was developed for the determination of indinavir concentrations in rat plasma, cerebrospinal fluid (CSF), and brain tissue homogenates. Biological samples were processed using a combination of acid pretreatment and liquid-liquid extraction with verapamil used as the internal standard. This method produced a linear response throughout the indinavir concentration range of 0.05-30 microM in plasma and 0.05-2.5 microM in CSF and brain with a LOD of 12.5 nM for plasma and CSF, and 6.25 nM for brain homogenate. Due to its high sensitivity, this assay is particularly useful for the quantitative determination of indinavir concentrations in brain and CSF.