Feasibility of a liquid-phase microextraction sample clean-up and liquid chromatographic/mass spectrometric screening method for selected anabolic steroid glucuronides in biological samples

Anabolic androgenic steroids (AAS) are metabolized extensively in the human body, resulting mainly in the formation of glucuronide conjugates. Current detection methods for AAS are based on gas chromatographic/mass spectrometric (GC/MS) analysis of the hydrolyzed steroid aglycones. These analyses require laborious sample preparation steps and are therefore time consuming. Our interest was to develop a rapid and straightforward method for intact steroid glucuronides in biological samples, using liquid-phase microextraction (LPME) sample clean-up and concentration method combined with liquid chromatographic/tandem mass spectrometric (LC/MS/MS) analysis. The applicability of LPME was optimized for 13 steroid glucuronides, and compared with conventional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) procedures. An LC/MS/MS method was developed for the quantitative detection of AAS glucuronides, using a deuterium-labeled steroid glucuronide as the internal standard. LPME, owing to its high specificity, was shown to be better suited than conventional LLE and SPE for the clean-up of urinary AAS glucuronides. The LPME/LC/MS/MS method was fast and reliable, offering acceptable reproducibility and linearity with detection limits in the range 2-20 ng ml(-1) for most of the selected AAS glucuronides. The method was successfully applied to in vitro metabolic studies, and also tested with an authentic forensic urine sample. For a urine matrix the method still has some unsolved problems with specificity, which should be overcome before the method can be reliably used for doping analysis, but still offering additional and complementary data for current GC/MS analyses.     

Analysis of steroid hormones in effluents of wastewater treatment plants by liquid chromatography-tandem mass spectrometry

This paper presents the development of an analytical procedure for the determination of two sexual steroid hormones: 17beta-estradiol and estrone, and the synthetic contraceptive estrogen, 17alpha-ethynylestradiol in effluents of wastewater treatment plants. Samples are extracted via solid-phase extraction using C18 cartridges. Extracts in ethyl acetate are then purified with a liquid-liquid separation with aqueous sodium chloride, then with a clean-up on a Florisil cartridge. Steroids are analyzed using an LC-MS-MS ion trap system. Internal quantification with the corresponding deuterated steroids leads to limits of quantification at 5 ng/l for estrone and 10 ng/l for estradiol and ethynylestradiol. In mineral spiked water, recoveries are 91% for 17beta-estradiol, 97% for estrone and 87% for 17alpha-ethynylestradiol and RSDs are 15% for 17beta-estradiol, 11% for estrone and 23% for 17alpha-ethynylestradiol.     

Determination of steroid hormones, hormone conjugates and macrolide antibiotics in influents and effluents of sewage treatment plants utilising high-performance liquid chromatography/tandem mass spectrometry with electrospray and atmospheric pressure chemical ionisation

In this study we present a high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method which has been elaborated to analyse steroid hormones, hormone conjugates, oral contraceptives and macrolide antibiotics unchanged in unfiltered influents and effluents of sewage treatment plants (STPs). HPLC separation of the steroid hormones was achieved in 35 min, as well as those of the antibiotics. The analytes were extracted by solid-phase extraction, followed by clean-up using size exclusion chromatography (SEC). For the final quantification HPLC/MS/MS was used. The two ionisation modes, electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI), in HPLC/MS/MS were compared for the analysis of steroid hormones. For quantitative results drastic matrix effects were observed while using ESI. These effects were less pronounced while using APCI. These pitfalls were additionally reduced by clean-up using SEC as well as isotope dilution. Additionally, two multiple reaction monitoring (MRM) transitions per compound were used to prevent false positive results. Recovery experiments with spiked tap water with concentrations varying from 1 to 1000 ng/L gave constant recovery rates: The recovery rates for the hormones and conjugates ranged from 58 to 107%, those of the contraceptives ranged from 83 to 109%. The relative standard deviation was found to be 7 to 24% and the limits of detection were 0.1 to 4.5 ng/L. The recovery rates of the macrolide antibiotics ranged from 76 to 103%, while the relative standard deviation was found to be 7 to 14% and the limits of detection ranged from 0.6 to 1.8 ng/L. The maximum concentrations found in influents of a STP was 470 ng/L for estriol and 1200 ng/L for erythromycin.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

A liquid chromatography–atmospheric pressure photoionization tandem mass spectrometric method for the determination of azaarenes in atmospheric particulate matter

The development, optimization and validation of a liquid chromatography–atmospheric pressure photoionization tandem mass spectrometric (LC–APPI/MS/MS) method for the determination of 15 azaarenes (4-azafluorene, benzo[h] and -[f]quinoline, phenanthridine, acridine, 1-azafluoranthene, 4-azapyrene, benz[a]- and -[c]acridine, -10-azabenzo[a]pyrene, 7,9- and 7,10-dimethylbenz[c]acridine, dibenz[a,j]-, -[c,h] and [a,i]acridine) in airborne particulate matter is described. Each compound was detected and quantified operating in multiple reaction monitoring mode. Extraction of azaarenes was achieved using accelerated solvent extraction (ASE) with dichlormethane/methanol (50/50, v/v). After extraction, no additional clean-up procedure like solid phase or liquid/liquid extraction was necessary. Limits of quantification (S/N × 10) ranged from 0.2 pg/μl to 1.4 pg/μl, matrix dependent recoveries were between 57% and 94%, with relative standard deviations from 8% to 17%. Applicability of the method was demonstrated analyzing 10 samples of particulate matter (PM_2.5) collected in winter 2008. In all samples dimethylbenz[c]acridines as well as dibenzacridines were below the limit of quantification, concentration of the remaining analytes were in the range from 0.002 ng/m³ to 0.356 ng/m³.     

Simple and sensitive liquid chromatographic quantitative analysis of the novel marine anticancer drug Yondelis (ET-743, trabectedin) in human plasma using column switching and tandem mass spectrometric detection

The development of a simple and sensitive assay for the quantitative analysis of the marine anticancer agent Yondelis (ET-743, trabectedin) in human plasma using liquid chromatography (LC) with column switching and tandem mass spectrometric (MS/MS) detection is described. After protein precipitation with methanol, diluted extracts were injected on to a small LC column (10 x 3.0 mm i.d.) for on-line concentration and further clean-up of the sample. Next, the analyte and deuterated internal standard were back-flushed on to an analytical column for separation and subsequent detection in an API 2000 triple-quadrupole mass spectrometer. The lower limit of quantitation was 0.05 ng mL(-1) using 100 micro l of plasma with a linear dynamic range up to 2.5 ng ml(-1). Validation of the method was performed according to the most recent FDA guidelines for bioanalytical method validation. The time needed for off-line sample preparation has been reduced 10-fold compared with an existing LC/MS/MS method for ET-743 in human plasma, employing a labor-intensive solid-phase extraction procedure for sample pretreatment. The proposed column switching method was successfully applied in phase II clinical trials with Yondelis and pharmacokinetic monitoring.     

The analysis of thyroxine in human serum by an 'exact matching' isotope dilution method with liquid chromatography/tandem mass spectrometry

A method for the analysis of thyroxine in human serum, utilising 'exact matching' isotope dilution mass spectrometry (IDMS) in combination with liquid chromatography/tandem mass spectrometry (LC/MS/MS), has been developed with a limit of quantification of 0.5 ng g(-1) of thyroxine in human serum. The extraction and clean-up of thyroxine from serum involves an efficient protein-precipitation stage followed by a solid-phase extraction procedure to produce an extract essentially free from interfering compounds. The method is reproducible, with expanded uncertainties of less than 2%, and is relatively fast to perform.     

Determination of endogenous and exogenous estrogens in effluents from sewage treatment plants at the ng/L-level

An analytical method for the determination of the major endogenous and exogenous estrogenic steriods in effluent water samples of sewage treatment plants (STPs) with a LOQ down to 1 ng/L and below has been developed. The exogenous estrogen 17α-ethynylestradiol, frequently used as estrogenic component in oral contraceptives, and the endogenous estrogen 17β-estradiol show the highest estrogenic potential, therefore they were part of our target compounds. In addition, the content of the synthetic gestagen levonorgestrel, also often administered in oral contraceptives, was determined. A solid-phase extraction system for high volume sampling of water up to 25 L was implemented. Two types of adsorbent, Amberlite XAD 2 and a mixture of LiChrolut EN/Bondesil C-18, respectively, were tested for their extraction efficiency of these polar analytes. Recovery rates with LiChrolut EN/¶Bondesil C-18 ranged up to 94 %, whereas sampling on XAD 2 led only to poor recoveries below 40 %. After a liquid chromatographic clean-up step on silicagel the steroids were converted into their trimethylsilyl-ethers by the reaction with MSTFA/TMSI (N-methyl-N-trimethylsilyl-2,2,2-trifluoroacetamide, trimethylsilyliodide) and were then determined by HRGC/MS in the selected ion mode. A limit of quantification over the whole procedure of at least 1 ng/L was reached for all analytes. In several effluent samples the input of estrogens by the STP of the cities Ulm and New Ulm into the river Danube was characterised. The concentrations commonly found ranged from 1 ng/L up to 13 ng/L, depending on the respective steroid.     

Liquid chromatography-tandem mass spectrometry analysis of estrogenic compounds in coastal surface water of the Baltic Sea

An analytical method has been developed for the determination of five naturally occurring estrogens (estradiol, estriol, estrone, genistein, daidzein), one synthetic hormone (ethynylestradiol) and three xenoestrogens (4-nonylphenol (NP), 4-tert-octylphenol (4-tert-OP), bisphenol A (BPA)) in coastal marine waters. The procedure includes a solid-phase extraction of approx. fifty litres of water samples on the solid-phase copolymer Oasis HLB followed by a clean-up on silica. Twenty-five percent aliquots were used for the analytical determination of the analytes using high performance liquid chromatography coupled with electrospray-ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Calculated extraction recoveries between 52 (4-tert-octylphenol) and 91% (nonylphenol) were obtained for the method developed. Matrix interferences occurring during electrospray ionisation were quantified by spiking the extracts prior to the measurements. Method detection limits ranged from 0.02 (estrone) to 1 ng L(-1) (estriol). The method was applied to determine environmental estrogens in coastal waters of the Baltic Sea. The analyses showed the presence of five compounds at levels between 0.10 (estrone) and 17 ng L(-1) (ethynylestradiol).     

Method development for the analysis of polybrominated dibenzo-p-dioxins, dibenzofurans and diphenyl ethers in sediment samples

A column chromatography procedure was developed for the clean-up of solvent-extracted sediment samples for the fractionation of polybrominated diphenyl ethers (PBDEs) and polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs). The procedure included multiple column chromatography steps for clean-up for the separation of PBDEs from PBDD/Fs. The separation of the two chemical groups overcame the mutual interfering problem during the GC-ion trap MS analysis. The method was validated with the analysis of quality control samples. The method accuracy represented with relative error was less than 16% for all targeted PBDEs and PBDD/Fs congeners. Recoveries of the ¹³C-labeled standards ranged from 64% to 117% with relative standard deviation from 7.3% to 15%. Results from the analysis of environmental sediment samples collected in the vicinity of a recycling site for electronic wastes showed high levels of PBDEs (1.5-12 ng/g, dry weight), trace levels of PBDFs (0.025-0.92 ng/g, dry weight) and non-detectable PBDDs.     

LC-MS/MS profiling for detection of endogenous steroids and prostaglandins in tissue samples

Roles of steroid hormones, and compounds that can influence their levels in cells, are of increasing interest in e.g. cancer research, partly because resistance to hormone therapies often complicates treatment. To elucidate the processes involved, the hormones and related compounds need to be accurately measured. Reversed-phase liquid chromatography with dynamic multiple reaction monitoring mass spectrometric detection in electrospray mode is capable of providing such measurements. Therefore, LC-MS/MS was developed for sensitive, selective analysis of 11 steroid hormones, cholesterol and two prostaglandins. The effects of the tissue matrix, and solid-phase extraction (SPE) sample clean-up, on the LC-MS/MS signals of the hormones were also investigated. The results show that the developed LC-MS/MS method, following SPE clean-up to reduce matrix interference, can detect selected steroids in extracts of mouse tissues. The method provides linear measurements of the steroids at concentrations up to few ng/μL, and limits of detection in the range 0.03-0.2 pg/μL (for some compounds lower than those of previously reported methods).     

Determination of steroid hormones in blood by GC–MS/MS

This paper presents the development, optimization and validation of a methodology to determine nine key steroid hormones (viz. pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, estrone, 17α-estradiol and 17β-estradiol) expressed in the steroidogenesis in biological fluids. The analytical method allows for the determination of steroid hormones in blood plasma and serum down to 0.08–0.16 ng/mL for estrogens, 0.20–0.36 ng/mL for androgens and 0.36–0.43 ng/mL for progestagens. These limits of detection were obtainable using a two-step solid-phase clean-up for fractionation and elimination of interfering lipids (fatty acids, phospholipids, glycerides and sterols) from the steroid hormones. The accuracy of the method was 50–112% in the range 0.10 to 2.00 ng/mL.     

Validation of a solid-phase extraction and liquid chromatography-electrospray tandem mass spectrometric method for the determination of nine basic pharmaceuticals in wastewater and surface water samples

This paper reports the development and validation of a quantitative LC-electrospray (ESI)-MS/MS method for the simultaneous analysis of nine basic pharmaceuticals (flubendazole, pipamperone, cinnarizine, ketoconazole, miconazole, rabeprazole, itraconazole, domperidone and propiconazole) in environmental waters. Sample preparation consisted of solid-phase extraction on a Speedisk phenyl and a NH(2) solid-phase extraction tube for sample clean-up. Chromatography was performed on a pentafluorophenyl column in a total run time of 24min. Due to different matrix effects measured in different surface water samples, standard addition was the only method to perform accurate quantification. Limits of detection and quantification were in the range of <0.05-1ng/l and 0.05-10ng/l, respectively. The method showed good precision and accuracy. Recoveries were in the range of 60-100%. This method allows to identify and quantify these pharmaceuticals in wastewater and surface water and enables to perform comprehensive studies on the occurrence in and removal of these drugs from influent and effluent wastewaters and surface waters.     

Combined isolation and purification procedures prior to the high-performance liquid chromatographic-ion-trap tandem mass spectrometric determination of estrogens and their conjugates in river sediments

A fast and sensitive analytical procedure has been developed for the simultaneous separation and determination of alpha-estradiol, beta-estradiol, estriol, estrone and ethynylestradiol and their sulfate, glucuronide and acetate conjugates in river sediments. The procedure includes a microwave-assisted extraction (MAE) with aqueous methanol (25:75, v/v) at 100 degrees C in 10 min, a clean-up on Oasis WAX cartridge and a high-performance liquid chromatography-ion-trap tandem mass spectrometry (HPLC-IT-MS/MS) with electrospray ionization. The recovery for each compounds ranged from 83 to 107% and the repeatability represented as RSDs ranged from 4.9 to 9.6%. The limits of detection (LODs) were down to 1 ng g(-1). The analytical performance of the method was evaluated using determination of free and conjugated estrogens in river sediments.     

UPLC–TOF–MS Method for Simultaneous Quantification of Steroid Hormones in Tissue Homogenates of Zebrafish with Solid-Phase Extraction

The quantification of steroid hormones of individual zebrafish (Danio rerio) provides perspective to understand endogenous hormone function. A UPLC–TOF–MS method was developed to provide a reproducible, sensitive, and efficient assay to determine the concentration of steroid hormones, including cortisol, testosterone, androstenedione, 11-deoxycortisol, 11-deoxycorticosterone, and 17-hydroxyprogesterone in whole-body homogenates of each zebrafish. Solid-phase extraction was used to sample matrix clean-up and acquired a recovery from 89.7% to 107.9%. The analytes were separated on an Aquity BEH C18 column using gradient elution. Mass spectrometric analysis was performed by single reaction monitoring (SRM) using positive electrospray ionization mode. The total running time was 6 min, which was greatly shortened compared with a previously reported method. The developed method exhibited excellent linearity for all the analytes, with regression coefficients higher than 0.99. The limit of detection varied between 0.1 and 0.5 ng/L and the limit of quantification was 0.5–1.7 ng/L for all analytes. The precision of the method was assessed on replicate measurements and was found to be in the ranges of 1.9 % to 6.6% and 4.3% to 8.6%, for intra- and inter-day analysis, respectively. This method was validated according to FDA guidance and applied to determine steroid hormone levels in the tissue homogenate of zebrafish acutely treated with caffeine and ethanol.     

Determination of selected human pharmaceutical compounds in effluent and surface water samples by high-performance liquid chromatography-electrospray tandem mass spectrometry

A simple method is presented for the analysis of 13 pharmaceutical and pharmaceutical metabolite compounds in sewage effluents and surface waters. The pharmaceutical compounds were extracted using a genetic solid-phase extraction (SPE) procedure using Phenomenex Strata X as a stationary phase. Extracts were quantitatively analysed by four separate reversed-phase high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) techniques and quantified by comparison with an internal standard ([13C]-phenacetin). Recoveries and limits of detection (LOD) for sulfamethoxazole (120%, 50 ng l(-1)), acetyl-sulfamethoxazole (56%, 50 ng l(-1)), trimethoprim (123%, 10 ng l(-1)), erythromycin (73%, 10 ng l(-1)), paracetamol (75%, 50 ng l(-1)), ibuprofen (117%, 20 ng l(-1)), clofibric acid (83%, 50 ng l(-1)), mefenamic acid (24%, 50 ng l(-1)), diclofenac (62%, 20 ng l(-1)), propranolol (45%, 10 ng l(-1)), dextropropoxyphene (63%, 20 ng l(-1)) and tamoxifen (42%, 10 ng l(-1)) were all acceptable. The recovery of lofepramine (4%) was too low to be of use in a monitoring programme. Application of the method to samples collected from UK sewage effluents and surface waters showed detectable concentrations of mefenamic acid, diclofenac, propranolol, erythromycin, trimethoprim and acetyl-sulfamethoxazole in both matrices. Ibuprofen and dextropropoxyphene were detected in sewage effluents alone. All other pharmaceutical compounds were below the methods limits of detection.     

Simultaneous determination of Delta9-tetrahydrocannabinol, 11-hydroxy-Delta9-tetrahydrocannabinol and 11-nor-9-carboxy- Delta9-tetrahydrocannabinol in human plasma by high-performance liquid chromatography/tandem mass spectrometry

A rapid and sensitive method for the simultaneous confirmatory analysis of three forensic most relevant cannabinoids, Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), by means of high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in human plasma was developed and fully validated. Sample clean-up was performed by automated silica-based solid-phase extraction and the separation was carried out using a PhenylHexyl column (50 x 2 mm i.d., 3 micro m) and acetonitrile-5 mM ammonium acetate gradient elution. Data were acquired with an API 3000 LC/MS/MS system equipped with a turboionspray interface and triple quadrupole mass analyzer using positive electrospray ionization and multiple reaction monitoring. Two MS/MS transitions for each substance were monitored and deuterated analogues of analytes were used as internal standards for quantitation. The limit of quantitation was 0.8 ng ml(-1) for THC, 0.8 ng ml(-1) for 11-OH-THC and 4.3 ng ml(-1) for THC-COOH and linearity with a correlation coefficient r(2) = 0.999 was achieved up to 100 ng ml(-1) for THC and 11-OH-THC and 500 ng ml(-1) for THC-COOH. The limits of detection were 0.2 ng ml(-1) for THC, 0.2 ng ml(-1) for 11-OH-THC and 1.6 ng ml(-1) for THC-COOH. The developed LC/MS/MS method was also successfully used for the determination of THC-COOH-glucuronide, the phase II metabolite of THC-COOH.     

Determination of gentamicin in swine and calf tissues by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used in veterinary medicine for the treatment of serious infections. The purpose of this study was to develop and validate a method to determine gentamicin residues in edible tissues of swine and calf. Extraction of gentamicin was performed using a liquid extraction with phosphate buffer containing trichloroacetic acid, followed by a solid-phase clean-up procedure on a CBA weak cation-exchange column. Tobramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS). Chromatographic separation of the internal standard tobramycin and the gentamicin components was achieved on a Nucleosil (5 microm) column using a mixture of 10 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. The gentamicin components C1a, C2 + C2a and C1 could be identified with the MS/MS detection, and subsequently quantified. The method was validated according to the requirements of the EC at the maximum residue limit (MRL) (100 ng g(-1) for muscle and fat, 200 ng g(-1) for liver and 1000 ng g(-1) for kidney), half the MRL and double the MRL levels. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit <10%). Limits of quantification of 25.0 ng g(-1) were obtained for the determination of gentamicin in muscle, fat, liver and kidney tissues of swine and calf, which correspond in all cases to at least half the MRLs. Limits of detection ranged between 0.5 and 2.5 ng g(-1) for the tissues. The within-day and between-day precisions (RSD) and the results for accuracy fell within the ranges specified. The method was successfully used for the determination of gentamicin in tissue samples of swines and calves medicated with gentamicin by intramuscular injection.     

Improved method for analyzing estrogens in water by liquid chromatography-electrospray mass spectrometry

An improved LC-electrospray ionization MS method was established for four estrogens (17beta-estradiol (E2), estriol (E3), estrone (E1), and ethynyl estradiol (EE)) in environmental water. Almost complete separation of all estrogens was achieved on a phenyl column with methanol/water as the mobile phase. Quantification was achieved in the negative ionization mode using selected ion monitoring. The instrumental detection limits were 20-30 ng/l for the four analytes. In Milli-Q spiked water, the recoveries of the four estrogens were 72-81%, which was similar to those found for river water spiked with the corresponding deuterated estrogens. The detection limits for the four estrogens in river water were 0.1-0.2 ng/l. The method was used to detect residual estrogens in the Tonghui River, which receives water from a municipal sewage treatment plant in Beijing; E1 (1.1 ng/l) and E2 (0.2 ng/l) were detected.     

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.