Automated Solid-phase Extraction Research Articles

Measurement of Total and Unbound Mycophenolic Acid and Its Glucuronide by LC Coupled with MS. Application to a Pharmacokinetic Study of Mycophenolate Mofetil in Patients with Autoimmune Diseases

Liquid chromatography coupled with mass spectrometry for the determination of total and unbound mycophenolic acid and its major metabolite in human plasma has been developed. Sample preparations were based on a fully automated solid-phase extraction process and ultrafiltration. Mass spectrometric data were acquired in a single-ion monitoring method. The analytes and nevirapine (internal standard) were well separated in an isocratic mode over 8 min. Validation study exhibited excellent linearity, with intra- and inter-day precision and accuracy of less than 12%. The assay was successfully applied to the pharmacokinetic study of mycophenolic acid in patients with autoimmune diseases.

Selection of SPE cartridge for automated solid-phase extraction of pesticides from water followed by liquid chromatography-tandem mass spectrometry

In environmental analyses there is an ever-increasing need to develop simple and sensitive multi-residue methods. In many agricultural regions, there is particular concern of the potential for pesticides to enter rivers and other waterways. This study reports on the development and validation of a multi-residue method of analysis for 30 pesticides in water samples using solid-phase extraction (SPE) followed by LC-MS/MS. The electrospray and MS/MS parameters were optimised for each pesticide, including capillary voltage, collision-induced dissociation voltage, and selection of a precursor ion and two product ions. A variety of SPE sorbents were tested for sample pre-concentration, including numerous polymeric based phases. Bond Elut PPL and Oasis HLB were the only phases capable of retaining the majority of the target analyte classes in a single method. An off-line pre-concentration method using a Gilson Aspec system was optimised using the Bond Elut PPL cartridges, with a concentration factor of 25 producing limits of quantitation in the order of 6-100 ng/L. Excellent linearity (r2 > 0.9), precision (<20%) and recovery (>60%) was obtained for nearly all of the analytes, covering a wide variety of chemical and physical properties. This is the first study to fully validate Bond Elut PPL cartridges for use in multi-residue pesticide analysis.

A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry

Measurement of 25-hydroxyvitamin D(3) (25OHD(3)) and D(2) (25OHD(2)) is challenging. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been described but they are often complex and difficult to automate. We have developed a simplified procedure involving an automated solid-phase extraction (SPE). Internal standard (hexadeuterated 25-hydroxyvitamin D(3)) was added to serum or plasma followed by protein precipitation with methanol. Following centrifugation, a robotic instrument (CTC PAL [Presearch] for ITSP SPE [MicroLiter Analytical Supplies, Inc]) performed a six-step SPE procedure and the purified samples were injected into the LC-MS/MS. Quantification of 25OHD(3) and 25OHD(2) was by electrospray ionization MS/MS in the multiple-reaction monitoring mode. The lower limit of quantitation was 4.0 nmol/L for 25OHD(3) and 7.5 nmol/L for 25OHD(2). Within- and between-assay precision was below 10% over the concentration range of 22.5-120 nmol/L for D(3) and 17.5-70 nmol/L for D(2) (n = 10). The calibration was linear up to 2500 nmol/L (r = 0.99). Recoveries ranged between 89% and 104% for both metabolites and no ion suppression was observed. The results obtained compared well (r = 0.96) with the IDS-OCTEIA 25-hydroxyvitamin D enzyme immunoassay for samples containing less than 125 nmol/L, at higher concentrations the immunodiagnostic system (IDS) method showed positive bias. Our simplified sample preparation and automated SPE method is suitable for the measurement of 25OHD(3) and D(2) in a routine laboratory environment. The system can process up to 300 samples per day with no cumbersome solvent evaporation step and minimal operator intervention.

Development of an automatic solid phase extraction and liquid chromatography mass spectrometry method by using a monolithic column for the analysis of Cyclosporin A in human plasma

A sensitive and specific and automated liquid chromatography–electrospray mass spectrometric (LC–ESI-MS) assay for the quantification of Cyclosporin A in human plasma was developed. Following a simple protein precipitation step, the supernatant was extracted on-line and directly injected into the system LC–ESI-MS. A relatively new type of monolithic column consisting of a silica rod with bimodal pore structure was used to achieve a retention time of 2.4 min with a very low backpressure at a flow rate of 1 ml/min. The assay was linear from 0.050 to 1.000 μg/ml. The mean recovery was 91%. The mean inter-day and intra-day precisions were 1.85% and 2.83%, respectively. The combination of the automated solid phase extraction and the low retention time achieved with this columns increase the throughput and decrease the time of analysis of each sample. This technology is useful in order to improve the efficiency of the bioanalytical studies.

Using total error concept for the validation of a liquid chromatography–tandem mass spectrometry method for the determination of budesonide epimers in human plasma

A robust, sensitive and selective method to quantify budesonide epimers in human plasma using solid-phase extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and fully validated. The drug was first isolated from the biological matrix by automated solid-phase extraction (SPE) on disposable extraction cartridges (C-2). The methanolic eluate was then collected and evaporated to dryness. The residue was dissolved in mobile phase and an aliquot was injected onto a Phenomenex Luna octadecylsilica (C-18) column (50 mm × 4.6 mm i.d., 3 μm). The mobile phase is composed of water containing 10 mM ammonium acetate adjusted to pH 3.2 with glacial acetic acid and acetonitrile (65:35, v/v). The flow-rate was 1.00 ml/min. Hydrocortisone acetate was used as internal standard (IS). Detection of the analytes was achieved using negative atmospheric pressure chemical ionization (APCI) tandem mass spectrometry in selected reaction monitoring (SRM) mode. The MS/MS ion transitions monitored were m/ z 489.3 → 357.3 and 463.3 → 403.2 for budesonide epimers and hydrocortisone, respectively. The method was validated using SFSTP (2003) proposal based on total measurement error and accuracy profiles as a decision tool. The most appropriate regression model for the response function as well as the limit of quantitation was first selected during the prevalidation step. These latter criteria were then assessed during the formal validation step. The limit of quantitation (LOQ) was around 50 pg/ml for budesonide epimers. The method was validated with respect to stability, recovery, linearity, precision, trueness and accuracy. Risk and uncertainty were also evaluated. The validated method was finally applied successfully to investigate the plasma concentration of budesonide epimers in a pharmacokinetic study.

Fully automatic method for the determination of fat soluble vitamins and vitamin D metabolites in serum

Background Fat soluble vitamins and vitamin D metabolites are key compounds in bone metabolism. Unfortunately, variability among 25(OH)D assays limits clinician ability to monitor vitamin D status, supplementation, and toxicity. Method 0.5 ml serum was mixed with 0.5 ml 60% acetonitrile 150 mM sodium dodecyl sulfate, vortexed for 30 s and injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration, then on-line transferred to a reversed-phase analytical column by a 15% methanol–acetonitrile mobile phase at 1.0 ml/min for individual separation of the target analytes. Ultraviolet detection was performed at 265 nm, 325 nm and 292 for vitamin D metabolites, vitamin A and α- and δ-tocopherols, respectively. Results Detection limits were between 0.0015 and 0.26 µg/ml for the target compounds, the precision (expressed as relative standard deviation) between 0.83 and 3.6% for repeatability and between 1.8 and 4.62% for within laboratory reproducibility. Recoveries between 97–100.2% and 95–99% were obtained for low and high concentrations of the target analytes in serum. The total analysis time was 20 min. Conclusions The on-line coupling of SPE-HPLC endows the proposed method with reliability, robustness, and user unattendance, making it a useful tool for high-throughput analysis in clinical and research laboratories.

Ultrasound-enhanced enzymatic hydrolysis of conjugated female steroids as pretreatment for their analysis by LC–MS/MS in urine

A fast, selective and sensitive method is here proposed for the analysis of female steroid hormones as conjugated forms (mainly, glucuronides and sulfates). The method has been applied to female urine samples to assess the metabolism of these compounds. The method implements an enzymatic hydrolysis (beta-glucuronidase with sulfatase activity) kinetically enhanced by ultrasonic energy in order to generate the free steroid forms. This enables a drastic shortening of the time required for this step as compared with conventional protocols (from 12-18 hours to 30 min). The reaction kinetics of the ultrasound-enhanced hydrolysis was characterized by comparison to that of the conventional protocol. After hydrolysis, the free steroid hormones were isolated and preconcentrated by automated solid-phase extraction and the eluate was subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The target analytes were confirmed and quantified by multiple reaction monitoring (MRM). The detection and quantification limits were within 0.06-0.8 ng mL(-1) and 0.19-2.69 ng mL(-1), respectively. The precision of the method, expressed as intra-day and inter-day variability, ranged between 2.1 and 5.2% and between 4.9 and 8.0%, respectively. A complementary study was carried out to assess the storage conditions of urine samples. This study is crucial in those applications involving metabolic processes as the integrity of the sample has to be preserved.

Automated Solid-Phase Extraction Approaches for Large Scale Biomonitoring Studies

The main value in measuring environmental chemicals in biological specimens (i.e., biomonitoring) is the ability to minimize risk assessment uncertainties. The collection of biomonitoring data for risk assessment requires the analysis of a statistically significant number of samples from subjects with a significant prevalence of detectable internal dose levels. This paper addresses the practical laboratory challenges that arise from these statistical requirements: development of high throughput techniques that can handle, with high accuracy and precision, a large number of samples and can do a trace level analysis of multiple and diverse environmental chemicals (i.e., analytes). We review here examples of high throughput, automated solid-phase extraction methods developed in our laboratory for biomonitoring of analytes with representative hydrophobic properties and for typical biomonitoring matrices. We discuss key aspects of sample preparation, column, and solvent selection for off- and online extractions, and the so-called nuts-and-bolts of online column-switching systems necessary for developing-with minimal sample handling-rugged, automated methods.

High through-put aflatoxin determination in plant material by automated solid-phase extraction on-line coupled to laser-induced fluorescence screening and determination by liquid chromatography–triple quadrupole mass spectrometry

A screening test based on laser-induced fluorescence (LIF) and a method for individual identification – quantitation of aflatoxins (AFs) in olive leaves and drupes, based on chromatographic separation and triple-quad mass-spectrometry detection with electrospray ionization in positive mode, is here reported. The sensitivity and selectivity of both methods are enhanced by a preconcentration–cleanup step developed by a Prospekt station. The analysis frequency is at least 3.5 samples/h. The screening test makes able to detect the target analytes at concentrations of 0.7 μg/kg without “false negatives”. The LC–MS/MS method provides limits of detection (LOD) and quantification (LOQ) ranging between 0.01–0.03 and 0.03–0.11 μg/kg, respectively. The linear dynamic range is between LOQ–50 μg/kg. The between-day precision, expressed as relative standard deviation, ranges between 0.97–2.86% and the within laboratory reproducibility, also expressed as RSD, between 1.63% and 4.84%.

Fullerenes for aromatic and non-aromatic N-nitrosamines discrimination

The detection of N-nitrosamines (NAms) in water supplies is an environmental and public health issue because many NAms are classified as probable human carcinogens. Non-aromatic (aliphatic and cyclic) NAms are more toxic than aromatic ones as their maximum admissible concentration is limited in drinking water (20–2000 ng L −1). From that premise, a simple and novel method to discriminate between both fractions of NAms according to their toxicity was proposed. An automatic solid-phase extraction unit containing two sequential sorbent columns was constructed. A sample volume of 25 mL was passed through a C 60 fullerene column in which only the aromatic fraction was retained, and the effluent was then passed through a Merck LiChrolut EN column where the non-aromatic fraction was retained. Following elution of the non-aromatic NAms with 150 μL of ethyl acetate–acetonitrile (9:1), 1 μL of the extract was injected into a GC/MS. A comparative study of C 60 and C 70 fullerenes and nanotubes revealed C 60 fullerene to be the best choice to selectively retain the aromatic fraction. The method exhibits a linear range of 15–20,000 ng L −1; limits of detection of 4–15 ng L −1; and an RSD of ∼5%. Recoveries throughout the whole method were between 95% and 102% for six non-aromatic NAms spiked into several types of waters. Our study demonstrates that a simple and fast SPE system (10 min per sample) with a customary GC–MS instrument permits the quantification of these amines in complex matrices with considerable sensitivity and selectivity.

Determination of Dialkyl Phosphate Metabolites of Organophosphorus Pesticides in Human Urine by Automated Solid-Phase Extraction, Derivatization, and Gas Chromatography-Mass Spectrometry

Organophosphorus (OP) pesticides are highly toxic but used commonly worldwide, nevertheless. Their urinary dialkylphosphate (DAP) metabolites are widely used for exposure assessment of OP pesticides in humans. We previously developed an analytical method to measure urinary DAPs utilizing solid-phase extraction (SPE)-derivatization-gas chromatography-tandem mass spectrometry (GC-MS-MS) with quantification using isotope-dilution technique. We now present a more cost-effective yet highly accurate method that can be easily adaptable to many laboratories for routine OP exposure assessment. This method is simple and fast and involves automated SPE of the metabolites followed by derivatization with pentafluorobenzyl bromide and quantification by GC-MS. Dibutyl phosphate (DBP) serves as the internal standard. The detection limits for the six metabolites ranged from 0.1 to 0.15 ng/mL. Depending on the metabolite the relative standard deviation of the analytical procedure was 2-15% for the metabolites. We compared performance of DBP as an internal standard with that of isotope-labeled compounds and found that DBP gives reliable results for the analytical procedure. We also optimized reaction parameters of pentafluorobenzylation.

Multisyringe flow injection system for solid-phase extraction coupled to liquid chromatography using monolithic column for screening of phenolic pollutants

In this work a fast, automatic solid-phase extraction procedure hyphenated to HPLC-UV is proposed for screening of priority phenolic pollutants in waters at ng mL −1 levels. A flow through column, containing polystyrene-divinylbenzene sorbent, was incorporated to a multisyringe flow injection system (MSFIA), where the sample loading and analyte elution were carried out after computer control. The MSFIA system also directed the eluent to fill the injection loop of the chromatograph, coupling the sample preparation to its determination. High enrichment factors were attained for phenol and ten of its derivatives (mean value 176 for 50 mL of sample), with LOD values lower than 1 ng mL −1 for the maximum volume of sample used (100 mL). For all analytes, mean recoveries between 89 and 103% were obtained for different water matrices. Certified reference material and a contaminated soil (RTC-CRM 112) were also tested successfully. The determination frequency was 4–10 h −1, providing an automatic, fast and reliable tool for water quality and environmental monitoring.

Automated solid-phase extraction for concentration and clean-up of female steroid hormones prior to liquid chromatography–electrospray ionization–tandem mass spectrometry: An approach to lipidomics

A method for determination of free and glucuronide-conjugated female steroid hormones in urine at the pg mL −1 level is here presented. For this purpose, a dual approach with or without β-glucuronidase hydrolysis has been developed to succeed in this analysis. The target analytes were two progestogens – progesterone and pregnenolone – and three endogenous estrogens – estradiol, estriol and estrone. Separation and detection were carried out by liquid chromatography electrospray ionization and tandem mass spectrometry (LC–ESI–MS–MS) with a triple quadrupole (qQq) mass detector. The determination step was optimized by multiple reaction monitoring for highly selective identification and sensitive quantification of female hormones in a complex sample such as human urine. As these compounds are present in urine at very low concentration (ng mL −1 level), a preconcentration and clean-up step by solid-phase extraction was automatically carried out prior to the chromatographic step in order to improve the sensitivity of the method. This sample pretreatment was performed using a lab-on-valve (LOV) manifold which provided preconcentration factors ranging from 59.1 to 72.3 for 10 mL urine. The detection and quantification limits were in the ranges 1.8–18 pg and 6–61 pg on-column, respectively, with precision values from 1.93 to 10.99%, expressed as relative standard deviation. These results enable to conclude the suitability of the LOV–LC–qQq approach for determination of the lipidomic profiling of the main female steroid hormones in a difficult matrix as human urine. The method can be potentially applied to the clinical and other metabolomic areas.

Automation of Solid-Phase Microextraction in High-Throughput Format and Applications to Drug Analysis

The automation of solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was accomplished using a 96 multiwell plate format, a SPME multifiber device, two orbital shakers, and a three-arm robotic system. Extensive optimization of the proposed setup was performed including coating selection, optimization of the fiber coating procedure, confirmation of uniform agitation in all wells, and the selection of the optimal calibration method. The system allows the use of pre-equilibrium extraction times with no deterioration in method precision due to reproducible timing of extraction and desorption steps and reproducible positioning of all fibers within the wells. The applicability of the system for the extraction of several common drugs is demonstrated. The optimized multifiber SPME-LC-MS/MS was subsequently fully validated for the high-throughput analysis of diazepam, lorazepam, nordiazepam, and oxazepam in human whole blood. The proposed method allowed the automated sample preparation of 96 samples in 100 min, which represents the highest throughput of any SPME technique to date, while achieving excellent accuracy (87-113%), precision (<or=20% RSD), and sensitivity (limit of quantitation 4 ng/mL). Automated SPME provides unique advantages over automated solid-phase extraction (SPE) including lower cost, the ability to quantitatively determine free and total drug concentrations in a single biofluid sample, and the ability to directly process whole blood samples with absolutely no sample pretreatment required.

Recent developments in automatic solid-phase extraction with renewable surfaces exploiting flow-based approaches

Solid-phase extraction (SPE) is the most versatile sample-processing method for removal of interfering species and/or analyte enrichment. Although significant advances have been made over the past two decades in automating the entire analytical protocol involving SPE via flow-injection approaches, on-line SPE assays performed in permanent mode lack sufficient reliability as a consequence of progressively tighter packing of the bead reactor, contamination of the solid surfaces and potential leakage of functional moieties. This article overviews the current state-of-the-art of an appealing tool for overcoming the above shortcomings, so-called bead-injection (BI) analysis, based on automated renewal of the sorbent material per assay exploiting the various generations of flow-injection analysis. It addresses novel instrumental developments for implementing BI and a number of alternatives for on-line chemical-derivatization reactions, and it pinpoints the most common instrumental detection techniques utilized. We present and discuss in detail relevant environmental and bioanalytical applications reported in the past few years.

LC–MS/MS method for the determination of nine antidepressants and some of their main metabolites in oral fluid and plasma: Study of correlation between venlafaxine concentrations in both matrices

In this paper, a fast, sensitive and selective LC–MS/MS method is described for the simultaneous determination of amitriptyline, imipramine, clomipramine, fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram and venlafaxine, as well as some of their main metabolites (nortriptyline, desipramine, norclomipramine and norfluoxetine), in oral fluid and plasma. The sample (0.2 mL) was extracted with an automated solid-phase extraction system (ASPEC XL), using mixed mode OASIS MCX cartridges. Chromatographic separation was performed in a Sunfire C18 IS column (20 mm × 2.1 mm, 3.5 μm), using a gradient of acetonitrile and ammonium formate (pH 3; 2 mM) as mobile phase, which allowed the elution of all the compounds in less than 5 min. The method has been fully validated in both specimens. This method was initially applied to the analysis of oral fluid and plasma samples from patients on antidepressant treatment in order to assess for which compounds it was likely to find a good correlation between both matrices. The best results were obtained for venlafaxine, so the study was extended for this compound, comparing the ratio between oral fluid and plasma concentrations ( R OF/PL) in five patients on venlafaxine treatment when both samples were collected simultaneously on four different occasions. An important inter and intraindividual variability was found in oral fluid concentrations for 150 mg dose (mean = 287.5 ng/m, range 58.8–531.2 ng/mL) and for 75 mg dose (mean = 186.3 ng/mL, range = 82.1–289.2 ng/mL). R OF/PL was calculated for each patient on the four different occasions, showing also a high variability (CV = 24.2–69.6%).

Analysis of nitrosamines in water by automated SPE and isotope dilution GC/HRMS: Occurrence in the different steps of a drinking water treatment plant, and in chlorinated samples from a reservoir and a sewage treatment plant effluent

A method based on automated solid-phase extraction (SPE) and isotope dilution gas chromatography/high resolution mass spectrometry (GC/HRMS) has been developed for the analysis of nine nitrosamines in water samples. The combination of automated SPE and GC/HRMS for the analysis of nitrosamines has not been reported previously. The method shows as advantages the selectivity and sensitivity of GC/HRMS analysis and the high efficiency of automated SPE with coconut charcoal EPA 521 cartridges. Low method detection limits (MDLs) were achieved, along with a greater facility of the procedure and less dependance on the operator with regard to the methods based on manual SPE. Quality requirements for isotope dilution-based methods were accomplished for most analysed nitrosamines, regarding to trueness (80–120%), method precision (<15%) and MDLs (0.08–1.7 ng/L). Nineteen water samples (16 samples from a drinking water treatment plant {DWTP}, 2 chlorinated samples from a sewage treatment plant {STP} effluent, and 1 chlorinated sample from a reservoir) were analysed. Concentrations of nitrosamines in the STP effluent were 309.4 and 730.2 ng/L, being higher when higher doses of chlorine were applied. N-Nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were the main compounds identified in the STP effluent, and NDEA was detected above 200 ng/L, regulatory level for NDMA in effluents stated in Ontario (Canada). Lower concentrations of nitrosamines were found in the reservoir (20.3 ng/L) and in the DWTP samples (n.d. −28.6 ng/L). NDMA and NDEA were respectively found in the reservoir and in treated and highly chlorinated DWTP samples at concentrations above 10 ng/L (guide value established in different countries). The highest concentrations of nitrosamines were found after chlorination and ozonation processes (ozonated, treated and highly chlorinated water) in DWTP samples.

Simultaneous Determination of Ketamine, Tramadol, Methadone, and their Metabolites in Urine by Gas Chromatography-Mass Spectrometry

An automated solid-phase extraction procedure combined with gas chromatography-mass spectrometry methodology, without derivatization, has been developed for the identification and quantitation of ketamine, norketamine, tramadol, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, and 2-ethyl-5-methyl-3,3-diphenylpyrroline in urine. The analytical approach is simple and rapid, yet reliable. Good linearity (r(2) > 0.995 over the concentration range of 30 to 600 ng/mL), sensitivity (limits of quantitation 15-30 ng/mL), accuracy (81.0-109.9%), precision (RSD < 13.8%), and recovery (> 79.6% in average) were achieved for all analytes. Ninety-one urine specimens from suspected drug users and 21 clinical urine specimens from methadone substitution therapy patients were analyzed to validate the method compatibility and stability. Results have demonstrated that this GC-MS method is a good confirmation and quantitation test scheme for the six target compounds in urine.

Concentrations of the sunscreen agent benzophenone-3 in residents of the United States: National Health and Nutrition Examination Survey 2003--2004.

BackgroundThe capability of benzophenone-3 (BP-3) to absorb and dissipate ultraviolet radiation facilitates its use as a sunscreen agent. BP-3 has other uses in many consumer products (e.g., as fragrance and flavor enhancer, photoinitiator, ultraviolet curing agent, polymerization inhibitor).ObjectivesOur goal was to assess exposure to BP-3 in a representative sample of the U.S. general population ≥ 6 years of age.MethodsUsing automated solid-phase extraction coupled to high-performance liquid chromatography–tandem mass spectrometry, we analyzed 2,517 urine samples collected as part of the 2003–2004 National Health and Nutrition Examination Survey.ResultsWe detected BP-3 in 96.8% of the samples. The geometric mean and 95th percentile concentrations were 22.9 μg/L (22.2 μg/g creatinine) and 1,040 μg/L (1,070 μg/g creatinine), respectively. Least-square geometric mean (LSGM) concentrations were significantly higher (p ≤ 0.04) for females than for males, regardless of age. LSGM concentrations were significantly higher for non-Hispanic whites than for non-Hispanic blacks (p ≤ 0.01), regardless of age. Females were more likely than males [adjusted odds ratio (OR) = 3.5; 95% confidence interval (95% CI), 1.9–6.5], and non-Hispanic whites were more likely than non-Hispanic blacks (adjusted OR = 6.8; 95% CI, 2.9–16.2) to have concentrations above the 95th percentile.ConclusionsExposure to BP-3 was prevalent in the general U.S. population during 2003–2004. Differences by sex and race/ethnicity probably reflect differences in use of personal care products containing BP-3.

An automated method for the simultaneous determination of pravastatin, 3-hydroxy isomeric metabolite, pravalactone and fenofibric acid in human plasma by sensitive liquid chromatography combined with diode array and tandem mass spectrometry detection

In this study, a sensitive and selective method based on liquid chromatography combined with diode array and tandem mass spectrometry detection (LC–DAD–MS/MS) was developed for the simultaneous quantitative determination of fenofibric acid, pravastatin and its main metabolites in human plasma. In this method, an automated solid-phase extraction (SPE) on disposable extraction cartridges (DECs) is used to isolate the compounds from the biological matrix and to prepare a cleaner sample before injection and analysis in the LC–DAD–MS/MS system. On-line LC–DAD–MS/MS system using an atmospheric pressure ionization (TurboIonSpray) was then developed for the simultaneous determination of pravastatin, 3-hydroxy isomeric metabolite (3-OH metab), pravalactone and fenofibric acid. The separation is obtained on an endcapped dodecyl silica based stationary phase using a mobile phase consisting of a mixture of acetonitrile, methanol and 5 mM ammonium acetate solution (30:30:40, v/v/v). Sulindac and triamcinolone were used as internal standards (ISs). The detection of the fenofibric acid and sulindac was achieved by means of a DAD system. The MS/MS ion transitions monitored were m/ z 442.2 → 269.1, 442.2 → 269.1, 424.3 → 183.0 and 435.2 → 397.2 for pravastatin, 3-OH metab, pravalactone and triamcinolone, respectively. The method was validated regarding stability, selectivity, extraction efficiency, response function, trueness, precision lower limit of quantitation and matrix effect. The limits of quantitation (LOQs) were around 0.50 ng/ml for pravastatin, 0.25 ng/ml for 3-OH metab, 0.05 ng/ml for pravalactone and 0.25 μg/ml for fenofibric acid.