Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography

The creatinine levels of blood and urine from humans, rats, and mice were measured by high-performance liquid chromatography. These were compared to the alkaline picrate analysis of creatinine performed by standard colorimetric, kinetic, and AutoAnalyzer techniques. For human serum and urine the values obtained using the HPLC technique gave good agreement with four out of five alkaline picrate techniques. For black or white mice, the serum creatinine concentration was 8.7 +/- 0.4 microM by HPLC but 44.9 +/- 1.9 microM by the lowest alkaline picrate method. Mouse urine creatinine concentrations were 3.24 +/- 0.19 mM by HPLC and 4.59 +/- 0.39 mM by the nearest alkaline picrate method. Rat serum creatinine concentrations analyzed by HPLC were about half the values obtained by AutoAnalyzer. Mouse and rat samples seemed to have substances which gave nonspecific color and thus interfered with the analysis of creatinine by the alkaline picrate methods. While the alkaline picrate analysis of creatinine was adequate for human samples, it was necessary to use HPLC to accurately measure rodent creatinine. The fractional excretion of creatinine was determined by measuring creatinine in mouse urine and plasma by both the kinetic and HPLC methods and comparing these values to urine and plasma inulin. Using the kinetic method, creatinine was cleared at 43 +/- 3% of the rate of inulin. Using the HPLC method, creatinine was cleared at 170 +/- 11% of the rate of inulin.     

One-step rapid extractive methylation of plasma nonesterified fatty acids for gas chromatographic analysis

The present report describes a one-step method for the derivatization and extraction of nonesterified fatty acids in plasma with subsequent analysis by conventional capillary gas-liquid chromatography or gas-liquid chromatography-mass spectrometry. The procedure requires 200 microliters of citrated plasma, dilution with 200 microliters of methanol containing a suitable internal standard, and rapid methylation (10 min) with ethereal diazomethane. An aliquot (60%) of the ether layer is subsequently removed, taken to dryness with nitrogen gas, and the residue is dissolved in a small volume of hexane (usually 50 microliters) for chromatographic analysis (taking 1 microliter for on-column injection). Samples are ready for analysis within 15 min after initial preparation of the plasma. The method has been found to be simple and rapid, providing clean fatty acid profiles. Although the method has been tested with 200 microliters of rat and human plasma, it can easily be adapted to a 40 microliters plasma sample if the esterified plasma extract is suspended in a smaller volume of hexane and/or a larger aliquot of the extract were to be injected into the gas chromatograph through use of a splitless injector.     

Direct analysis of salicylic acid,salicyl acyl glucuronide,salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C₁₈ column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3–4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2–20 μg/ml and linearity was observed from 0.1–200 μg/ml and 5–2000 μg/ml for SA in plasma and urine, respectively. The method was validated to 0.2 μg/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.     

The simple and sensitive measurement of malondialdehyde in selected specimens of biological origin and some feed by reversed phase high performance liquid chromatography

A method for the determination of malondialdehyde (MDA) concentrations in specimens of animal tissues and feed has been developed using high performance liquid chromatography. The MDA concentration in acidified urine samples was determined after its conversion with 2,4-dinitrophenylhydrazine (DNPH) to a hydrazone (MDA-DNPH). Samples of blood plasma, muscle, liver and feed were prepared by saponification followed by derivatisation with DNPH to MDA-DNPH. The MDA concentration in chicken and hen feed samples was analysed after saponification and derivatisation followed by extractions with hexane. The free MDA in plasma samples was determined after deproteinization followed by derivatisation of MDA with DNPH. The chromatographic separation of MDA-DNPH samples was conducted using Phenomenex C(18)-columns (Synergi 2.5 μm, Hydro-RP, 100 ?, the length of 100mm) with an inner diameter of 2 or 3mm. MDA in processed biological samples was analysed using a linear gradient of acetonitrile in water, and the photodiode detector was set to 307 or 303 nm for detection. The current method that was utilised was based on the high-efficient derivatisation of MDA and was more sensitive compared to previously used methods. The selective and sensitive photodetection of the column effluent was found to be suitable for the routine analysis of MDA in urine, plasma, muscles and liver of animals and some feed samples. Because urine or blood plasma samples can be derivatised in a simple manner, the proposed method can also be suitable for the routine, non-invasive evaluation of oxidative stress in animals and humans.     

Determination of iothalamate in rat urine,plasma, and tubular fluid by capillary electrophoresis

A method for the quantitative determination of iothalamate (IOT) in rat urine, plasma and tubular fluid by capillary zone electrophoresis (CE) has been developed and validated. Samples of urine and tubular fluids were diluted with water and samples of plasma were deproteinized with two volumes of acetonitrile containing the internal standard, p-aminobenzoic acid (PABA). A BioFocus 2000 system (Bio-Rad, Hercules, CA, USA) was used. The UV detector was set at 254 nm. The samples were loaded into uncoated fused-silica capillary (40 cm×50 μm) by pressure injection. A borate buffer [20 mM, pH 12 (pH adjusted with 1.0 M NaOH)] was used as the electrophoretic buffer. The typical analytical conditions were: voltage, 22 kV; injection, 9 psi×s; capillary and carousel temperatures were 20°C and 18°C respectively. The linear relationship was observed between time-corrected peak area of IOT in water and urine or the corrected peak area ratio of IOT to PABA in plasma and the nominal concentration of IOT with correlation coefficient greater than 0.999. The intra- and inter-day coefficients of variation (CV) were less than 8%. The concentration of IOT in plasma, urine and tubular fluid determined by CE can be used for estimation of whole kidney and single nephron clearances.     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

Determination of beta-phenylethylamine concentrations in human plasma, platelets, and urine and in animal tissues by high-performance liquid chromatography with fluorometric detection

A highly sensitive method for the determination of beta-phenylethylamine in human plasma, platelets, and urine and in mouse tissue is described. The method is based on a two-step isolation using cation-exchange columns followed by reverse phase high-performance liquid chromatography with fluorometric detection. The recovery of the amine through the whole procedure was almost complete, ranging from 99 to 101%. The calibration graph appeared linear over the range of 50 to 5000 pg/injection. Urinary excretion of beta-phenylethylamine in humans ranged from 0.93 to 51.20 ng/mg creatinine. The amine was also detectable in plasma and platelets. Of the various mouse tissues examined, the highest concentrations were found in the small intestine, followed by the blood and liver. Concentrations of about 5 ng/g wet wt were detected in brain tissue, which increased remarkably after inhibition of monoamine oxidase by pargyline.     

Quantification of acetylcholine, choline, betaine, and dimethylglycine in human plasma and urine using stable-isotope dilution ultra performance liquid chromatography-tandem mass spectrometry

Disorders in choline metabolism are related to disease conditions. We developed a stable-isotope dilution ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of acetylcholine (ACh), betaine, choline, and dimethylglycine (DMG). We used this method to measure concentrations of the analytes in plasma and urine in addition to other biological fluids after a protein precipitation by acetonitrile. The detection limits were between 0.35 nmol/L (for ACh in urine) and 0.34 μmol/L (for betaine in urine). ACh concentrations were not detectable in plasma. Intraassay and interassay coefficient of variation (CVs) were all <10.0% in biological fluids, except for DMG in cerebrospinal fluid (CV=12.44%). Mean recoveries in urine pool samples were between 99.2% and 103.9%. The urinary excretion of betaine, choline, and DMG was low, with approximately 50.0% higher excretion of choline in females compared to males. Median urinary excretion of ACh were 3.44 and 3.92 μmol/mol creatinine in males and females, respectively (p=0.689). Plasma betaine concentrations correlated significantly with urinary excretions of betaine (r=0.495, p=0.027) and choline (r=0.502, p=0.024) in females. Plasma choline concentrations correlated significantly with urinary excretion of ACh in males (r=0.419, p=0.041) and females (r=0.621, p=0.003). The new method for the simultaneous determination of ACh, betaine, choline, and DMG is sensitive, precise, and fast enough to be used in clinical investigations related to the methylation pathway.     

LC/MS/MS measurement of penicillin G in bovine plasma, urine, and biopsy samples taken from kidneys of standing animals

Methods for the measurement of penicillin concentration in bovine plasma, kidney and urine were developed and validated. Detection was based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Phenethecillin was used as an internal standard. Plasma was extracted with acetonitrile using a method with a calculated limit of quantitation (LOQ) of 12 ng/mL. Kidney samples were homogenized in water and acetonitrile, then cleaned up on C18-bonded silica SPE cartridges. The LOQ of this procedure was 10 ng/g. Urine samples were diluted, filtered, and analyzed directly. The LOQ of this procedure was 63 ng/mL. The overall accuracy for plasma was 103% with coefficient of variation (CV) of 3%; for kidney, 96% and 11%, respectively, and for urine, 98% and 4%, respectively. These methods were applied to the analysis of plasma, urine, and kidney biopsy samples taken from standing animals that had been dosed with penicillin.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

High-performance liquid chromatographic method for determination of amodiaquine,chloroquine and their monodesethyl metabolites in biological samples

A high-performance liquid chromatographic method for determination of amodiaquine (AQ), desethylamodiaquine (DAQ), chloroquine (CQ) and desethylchloroquine (DCQ) in human whole blood, plasma and urine is reported. 4-(4-Dimethylamino-1-methylbutylamino)-7-chloroquinoline was used as internal standard. The drugs and the internal standard were extracted into di-isopropyl ether as bases and then re-extracted into an acidic aqueous phase with 0.1 M phosphate buffer at pH 4.0 for AQ samples and at pH 2.5 for CQ filter paper samples. A C(18) column was used and the mobile phase consisted of methanol-phosphate buffer (0.1 M, pH 3)-perchloric acid (250: 747.5:2.5, v/v). The absorbance of the drugs was monitored at 333 nm and no endogenous compound interfered at this wavelength. The limit of quantification in whole blood, plasma and urine was 100 nM for AQ and DAQ (sample size 100 microliter) as well as for CQ and DCQ in blood samples dried on filter paper. For 1000 microliter AQ and DAQ samples, the limit of quantification was 10 nM in all three biological fluids. The within-assay and between-assay coefficients of variations were always <10% at the limits of quantification. Plasma should be preferred for the determination of AQ and DAQ since use of whole blood may be associated with stability problems.     

Oxidative DNA damage in vivo: relationship to age, plasma antioxidants, drug metabolism, glutathione-S-transferase activity and urinary creatinine excretion

Oxidative DNA modification has been implicated in development of certain cancers and 8-oxodG, the most abundant and mutagenic DNA modification, has for some time been considered a biomarker of this activity. Urinary excretion of 8-oxodG over 24h has been used to estimate the rate of damage to DNA, and animal studies have supported this rationale. Reported determinants include tobacco smoking, heavy exercise, environmental pollution and individual oxygen consumption. Samples from three published studies were used to determine the association of urinary 8-oxodG excretion with age, plasma antioxidants, the glutathione-S-transferase phenotype and the activity of the xenobiotic metabolising enzyme CYP1A2. In the age range 35-65 years, age was not related to urinary 8-oxodG excretion, and there were no relations to either the glutathione-S-transferase phenotype or to the plasma antioxidants: vitamin C, alpha-tocopherol, beta-carotene, lycopene or coenzyme Q10. The activity of CYP1A2 showed a significant correlation in two of the three studies, as well as a significant correlation of 0.26 (p < 0.05) in the pooled data set. Regression analysis of CYP1A2 activity on 8-oxodG indicated that 33% increase in CYP1A2 activity would correspond to a doubling of 8-oxodG excretion. This finding needs to be confirmed in independent experiments. Spot morning urine samples can under certain circumstances be used to estimate 8-oxodG excretion rate provided that creatinine excretion is unchanged (in paired experiments) or comparable (in un-paired experiments), as evaluated from the correlation between 8-oxodG excretion in 24 h urine samples and in morning spot urine samples corrected for creatinine excretion (r = 0.50, p < 0.05). We conclude that 8-oxodG excretion is determined by factors like oxygen consumption and CYP1A2 activity rather than by factors like plasma antioxidant concentrations.     

Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets,erythrocytes, and leukocytes

Increasing demand for quality control of blood products requires more sensitive methods to enumerate residual cells. Presently, the reported threshold (in cells per microliter) is 400 for red blood cells, 30-500 for platelets, and 1 for leukocytes. To examine precision and linearity in enumerating residual platelets and red blood cells, EDTA-anticoagulated blood from healthy donors was serially diluted with serum, stained in TruCount tubes using a no-lyse/no-wash procedure and a monoclonal antibody cocktail against the CD42a (FL1) and glycophorin-A (FL2) epitopes, and analyzed by flow cytometry. Leukocyte counts were determined in separate tubes. Cell preparation and analysis were performed once for 20 blood samples each and 20 times using the same specimen. Acquisition from the same tube was performed separately for platelets (threshold on FL1) and red blood cells (threshold on FL2). Multiparameter analysis was used for data evaluation. Linear results were obtained for platelets per microliter between 3,410 and 5 and for red blood cells per microliter between 54,000 and 3. For the lower cell concentrations, the coefficient of variation was 16.7% for platelets and 10.9% for red blood cells. The presented method allows the distinction between physiologically intact and ghost red blood cells. The method represents a reliable, sensitive, and accurate approach to quantify platelets and red blood cells in diluted blood. It can be applied to enumerate residual cells in plasma products and meets the increasing demand for quality control in blood components.     

Determination of carboplatin in plasma and tumor by high-performance liquid chromatography-mass spectrometry

Carboplatin is a platinum analogue that is used in a number of chemotherapeutic regimens for solid tumors, such as lung and ovarian carcinomas. Most often characterization of carboplatin's pharmacokinetic properties is based on measurement of platinum, rather than intact carboplatin. We have developed a sensitive LC-MS method for the determination of intact carboplatin in plasma ultrafiltrate and in tumor tissue. Carboplatin was extracted from rat plasma ultrafiltrate and tumor samples using solid-phase extraction cartridges and analyzed using reversed-phase chromatography with positive electrospray ionization followed by mass spectrometric detection. Using 50 microliter of plasma ultrafiltrate or 140 microliter of tumor homogenate supernatant, the extraction afforded a recovery of 58.7 and 45.8% for plasma and tumor, respectively. The mobile phase was 5% acetonitrile in 0.5% acetic acid at 0.2 ml/min that yielded a retention time of carboplatin of 2.2 min. The method has been validated at carboplatin plasma ultrafiltrate concentrations from 0.07 to 2.5 microgram/ml, and from 0.03 to 1.3 microgram/ml in tumor homogenates. The main advantages of this method compared with earlier methods are the ability to measure intact carboplatin in a sensitive and specific manner.     

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.     

Analysis of creatine, creatinine, creatine-d3 and creatinine-d3 in urine, plasma, and red blood cells by HPLC and GC-MS to follow the fate of ingested creatine-d3

Creatine, which is increasingly being used as an oral supplement, is naturally present in the body. Studies on the fate of a particular dose of creatine require that the creatine be labeled, and for studies in humans the use of a stable isotopic label is desirable. The concentrations of total creatine and total creatinine were determined using HPLC. Creatine and creatinine were then separated using cation exchange chromatography and each fraction was derivatized with trifluoroacetic anhydride and the ratio of the deuterated:undeuterated species determined using GC-MS. Ratios of creatine:creatine-d(3), and creatinine:creatinine-d(3), and the concentrations of each of these species, were able to be determined in urine, plasma and red blood cells. Thus, the uptake of labeled creatine into plasma and red blood cells and its excretion in urine could be followed for a subject who ingested creatine-d(3). Creatine-d(3) was found in the plasma and red blood cells 10 min after ingestion, while creatine-d(3) and creatinine-d(3) were found in the urine collected after the first hour.     

Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes

Methadone and buprenorphine are two of the drugs most frequently used for abstinence from illicit opioids and in the treatment of pain. A sensitive and selective high-performance liquid chromatographic method with diode array detection for the simultaneous determination of methadone, buprenorphine and norbuprenorphine has been developed. Separation of the three analytes was obtained by using a reversed-phase column (C8, 250mmx4.6mm i.d., 5microm) and a mobile phase composed of 40% phosphate buffer containing triethylamine, 50% methanol and 10% acetonitrile (final apparent pH 6.0). Loxapine was used as the internal standard. An accurate pre-treatment procedure of biological samples was developed, using solid-phase extraction with C8 cartridges (100mg, 1mL) and needing small amounts of plasma or urine (300microL). The calibration curves were linear over a working range of 10.0-1500.0ng/mL for methadone and of 5.0-500.0ng/mL for buprenorphine and norbuprenorphine in both matrices. The limit of quantitation (LOQ) and the limit of detection (LOD) were 1.0 and 0.4ng/mL for methadone and 0.5 and 0.2ng/mL for both buprenorphine and norbuprenorphine, respectively. The method was successfully applied to the analysis of plasma and urine samples from patients undergoing treatment with these drugs. Precision and accuracy results were satisfactory and no interference from endogenous or exogenous compounds was found. The method is suitable for the simultaneous determination of methadone and buprenorphine in human plasma and urine for therapeutic drug monitoring purposes.     

Colorimetric plasma assay for the bentiromide test (BT-PABA) for exocrine pancreatic insufficiency

Bentiromide is a synthetic peptide, N-benzoyl-L-tyrosyl-p-aminobenzoic acid, which has been used as a test for exocrine pancreatic function. Following oral administration, bentiromide is hydrolyzed by chymotrypsin to yield free p-aminobenzoic acid (PABA) which is absorbed, conjugated and excreted in the urine. The PABA conjugates reach their peak levels in blood in 90-120 min. Healthy individuals have higher levels of PABA than patients with pancreatic insufficiency. A simple, accurate, and precise method for the determination of PABA in blood has been developed and validated. The plasma (1 ml) is deproteinized by perchloric acid. The conjugates are hydrolyzed and the total PABA is determined colorimetrically by the Bratton-Marshall test. The standard curve in plasma is linear up to 8 micrograms/ml of PABA. A similar semimicro method using 200 microliter of plasma suitable for pediatric samples shows comparable results. Average analytical recovery is 97% and precision studies of pooled within-run and total between-run showed CV% of 5.0 and 5.7%, respectively.     

Determination of azosemide and its metabolite in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the determination of azosemide and its metabolite, M1, in human plasma and urine and rabbit blood and tissue homogenates. The methods involved deproteinization of the biological samples: 2.5 volumes of acetonitrile were used for the determination of azosemide and 1 volume of saturated Ba(OH)₂ and ZnSO₄ for that of M1. A 50-μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column in each instance. The mobile phases employed were 0.03 M phosphoric acid—acetonitrile (50:40, v/v) for azosemide and 0.03 M phosphoric acid/0.2 M acetic acid—acetonitrile (83:17, v/v) for M1. The flow-rate was 1.5 ml/min in both instances. The column effluent was monitored by ultraviolet detection at 240 and 236 nm for azosemide and M1, respectively. The retention times for azosemide and M1 were 6.0 and 8.3 min, respectively. The detection limits for both azosemide and M1 in both human plasma and urine were 50 ng/ml. The coefficients of variation of the assay were generally low (below 11.0%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or other diuretics tested were observed.     

Analysis of risperidone and 9-hydroxyrisperidone in human plasma, urine and saliva by MEPS-LC-UV

Risperidone is currently one of the most frequently prescribed atypical antipsychotic drugs; its main active metabolite 9-hydroxyrisperidone contributes significantly to the therapeutic effects observed. An original analytical method is presented for the simultaneous analysis of risperidone and the metabolite in plasma, urine and saliva by high-performance liquid chromatography coupled to an original sample pre-treatment procedure based on micro-extraction by packed sorbent (MEPS). The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 73% (v/v) acidic phosphate buffer (30 mM, pH 3.0) containing 0.23% triethylamine and 27% (v/v) acetonitrile. The UV detector was set at 238 nm and diphenhydramine was used as the internal standard. The sample pre-treatment by MEPS was carried out on a C8 sorbent. The extraction yields values were higher than 92% for risperidone and 90% for 9-hydroxyrisperidone, with RSD for precision always lower than 7.9% for both analytes. Limit of quantification values in the different matrices were 4 ng/mL or lower for risperidone and 6 ng/mL or lower for the metabolite. The method was successfully applied to plasma, urine and saliva samples from psychotic patients undergoing therapy with risperidone, with satisfactory accuracy results (recovery>89%) and no interference from other drugs. Thus, the method seems to be suitable for the therapeutic drug monitoring of schizophrenic patients using the three different biological matrices plasma, urine and saliva.