Mohamed Abdel-Rehim

Recent advances in microextraction by packed sorbent for bioanalysis

Microextraction by packed sorbent (MEPS) is a new format for solid-phase extraction (SPE) that has been miniaturized to work with sample volumes as small as 10muL. The commercially available presentation of MEPS uses the same sorbents as conventional SPE columns and so is suitable for use with most existing methods by scaling the reagent and sample volumes. Unlike conventional SPE columns, the MEPS sorbent bed is integrated into a liquid handling syringe that allows for low void volume sample manipulations either manually or in combination with laboratory robotics. The key aspect of MEPS is that the solvent volume used for the elution of the analytes is of a suitable order of magnitude to be injected directly into GC or LC systems. This new technique is very promising because it is fast, simple and it requires very small volume of samples to produce comparable results to conventional SPE technique. Furthermore, this technique can be easily interfaced to LC/MS and GC/MS to provide a completely automated MEPS/LC/MS or MEPS/GC/MS system. This extraction technique (MEPS) could be of interest in clinical, forensic toxicology and environmental analysis areas. This review provides a short overview of recent applications of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine. The extraction of anti-cancer drugs, beta-blockers drugs, local anaesthetics, neurotransmitters and antibiotics from biological samples using MEPS technique will be illustrated.

Microextraction by packed sorbent (MEPS) : A tutorial

This tutorial provides an overview on a new technique for sample preparation, microextraction by packed sorbent (MEPS). Not only the automation process by MEPS is the advantage but also the much smaller volumes of the samples, solvents and dead volumes in the system. Other significant advantages such as the speed and the simplicity of the sample preparation process are provided. In this tutorial the main concepts of MEPS will be elucidated. Different practical aspects in MEPS are addressed. The factors affecting MEPS performance will be discussed. The application of MEPS in clinical and pre-clinical studies for quantification of drugs and metabolites in blood, plasma and urine will be provided. A comparison between MEPS and other extraction techniques such as SPE, LLE, SPME and SBSE will be discussed.

Screening of Cocaine and Its Metabolites in Human Urine Samples by Direct Analysis in Real-Time Source Coupled to Time-of-Flight Mass Spectrometry After Online Preconcentration Utilizing Microextraction by Packed Sorbent

Microextraction by packed sorbent (MEPS) has been evaluated for fast screening of drugs of abuse with mass spectrometric detection. In this study, C8 (octyl-silica, useful for nonpolar to moderately polar compounds), ENV+ (hydroxylated polystyrene-divinylbenzene copolymer, for extraction of aliphatic and aromatic polar compounds), Oasis MCX (sulfonic-poly(divinylbenzene-co-N-polyvinyl-pyrrolidone) copolymer), and Clean Screen DAU (mixed mode, ion exchanger for acidic and basic compounds) were used as sorbents for the MEPS. The focus was on fast extraction and preconcentration of the drugs with rapid analysis using a time-of-flight (TOF) mass spectrometer as the detector with direct analysis in a real-time (DART) source. The combination of an analysis time of less than 1 min and accurate mass of the first monoisotopic peak of the analyte and the relative abundances of the peaks in the isotopic clusters provided reliable information for identification. Furthermore, the study sought to demonstrate that it is possible to quantify the analyte of interest using a DART source when an internal standard is used. Of all the sorbents used in the study, Clean Screen DAU performed best for extraction of the analytes from urine. Using Clean Screen DAU to extract spiked samples containing the drugs, linearity was demonstrated for ecgonine methyl ester, benzoylecgonine, cocaine, and cocaethylene with average ranges of: 65–910, 75–1100, 95–1200, and 75–1100 ng/mL (n = 5), respectively. The limits of detection (LOD) for ecgonine methyl ester, benzoylecgonine, cocaine, and cocaethylene were 22. 9 ng/mL, 23. 7 ng/mL, 4. 0 ng/mL, and 9.8 ng/mL respectively, using a signal-to-noise ratio of 3:1.

The effect of busulphan on the pharmacokinetics of cyclophosphamide and its 4-hydroxy metabolite: time interval influence on therapeutic efficacy and therapy-related toxicity

Busulphan and cyclophosphamide (Bu/CP) are widely used in preparative regimens for bone marrow transplantation. Many studies have shown a wide variation in busulphan pharmacokinetics. Moreover, higher rates of liver toxicity were reported in Bu/CP protocols than in a total body irradiation (TBI)-containing regimen. In the present paper we investigated the effect of the time interval between the last dose of busulphan and the first dose of cyclophosphamide on the pharmacokinetics of CP and its cytotoxic metabolite 4-hydroperoxycyclophosphamide (4-OHCP). Thirty-six patients undergoing bone marrow transplantation (BMT) were included in the study. We also investigated the occurrence of veno-occlusive disease, mucositis and graft-versus-host disease. Ten patients conditioned with CP followed by TBI served as a control group (TBI). Twenty-six patients were conditioned with Bu/CP. The patients received Bu (1 mg/kg × 4 for 4 days), followed by CP (60 mg/kg for 2 days) administered as a 1-h infusion. Patients received their CP therapy either 7–15 h (group A, n = 12) or 24–50 h (group B, n = 14) after the last dose of Bu. None of the patients were given phenytoin or any other drug known to enhance CP metabolism. The administration of CP less than 24 h after the last dose of Bu resulted in: (1) a significantly (P = 0.003) lower clearance for cyclophosphamide was observed in group A (0.036 l/h/kg) compared to 0.055 and 0.055 l/h/kg, in the B and TBI groups, respectively; (2) significantly (P = 0.002) longer elimination half-life in group A (10.93 h) than in groups B and TBI (6.87 and 7.52 h, respectively); (3) significantly (P < 0.001) lower exposure to the cytotoxic metabolite (4-ohcp), expressed as the ratio auc4-OHCP/AUCCP, in group A (0.0053) than that obtained in group B (0.013) and group TBI (0.012); (4) the patients in group A had a significantly (P < 0.05) higher incidence of vod (seven of 12) than the other groups, b and tbi (2/14 and 1/10, respectively); and (5) mucositis was significantly higher in group a patients (8/12), being seen in only one patient in group b and none in the tbi group. the present study has shown that the interval between busulphan and cyclophosphamide administration can negatively affect the pharmacokinetics of cyclophosphamide and its cytotoxic metabolite. we conclude that the timing of cp administration must be considered in order to improve drug efficacy and reduce conditioning-related toxicity. Bone Marrow Transplantation (2000) 25, 915–924.

Study of the factors affecting the performance of microextraction by packed sorbent (MEPS) using liquid scintillation counter and liquid chromatography-tandem mass spectrometry

Microextraction by packed sorbent (MEPS) is a new technique for sample preparation that can be connected on-line with LC or GC. In MEPS, approximately 1-2mg of the solid packing material is inserted into a syringe (100-250 microL) as a plug. Sample preparation takes place on the packed bed. The bed can be packed or coated to provide selective and suitable sampling conditions. The new method is very promising for extraction of drugs and metabolites from biological samples. In this paper, some factors affecting the performance of MEPS such as recovery, carry-over, leakage, washing volume and elution volume were studied using C18 and hydroxylated polystyrene-divinylbenzene copolymer (ENV+) as sorbents. Radioactively labelled bupivacaine in plasma samples was used as test analyte. For the extraction of this drug, using methanol/water 95:5 (v/v) (0.25% ammonium hydroxide) was used as elution solvent. The analyte response increased with increasing the elution volume and it was linear upp up to 100 microL utilizing liquid scintillation counter. Further, for concentrating the sample, we found that MEPS may be used such that the sample can be drawn through the needle, up and down, several times. The analyte leakage increases as the volume washing increases, though higher washing volumes may also result in cleaner extracts. To eliminate analyte carry-over, the sorbents were washed first with 3 x 250 microL elution solution and then with 3 x 250 microL washing solution. In addition, the reproducibility measurements show relatively good relative standard deviation (RSD) % values concerning analyte recovery and analyte leakage. The present study provides an understanding of basic aspects when optimizing methods for MEPS. In this study, MEPS was used off-line with liquid scintillation counter and on-line with LC-MS/MS.

Microextraction in Packed Syringe Online with Liquid Chromatography‐Tandem Mass Spectrometry: Molecularly Imprinted Polymer as Packing Material for MEPS in Selective Extraction of Ropivacaine from Plasma

Abstract The excellent performance of a new sample preparation method, microextraction in packed syringe (MEPS), was recently illustrated by online LC‐MS and GS‐MS assays of local anaesthetics in plasma samples. In the method, approximately 1 mg of solid packing material was inserted into a syringe (100–250 µL) as a plug. Sample preparation took place on the packed bed. The new method was easy to use, fully automated, of low cost, and rapid in comparison with previously used methods. This paper presents the use of molecularly imprinted polymers (MIPs) as packing material for higher extraction selectivity. Development and validation of a method for MIP‐MEPS online with LC‐MS‐MS using ropivacaine in plasma as model compound were investigated. A bupivacaine imprinted polymer was used. The method was validated and the standard curves were evaluated by means of quadratic regression and weighted by inverse of the concentration: 1/x for the calibration range 2–2000 nM. The applied polymer could be used more than 100 times before the syringe was discarded. The extraction recovery was 60%. The results showed high correlation coefficients (R2>0.999) for all runs. The accuracy, given as a percentage deviation from the nominal concentration values, ranged from −6% to 3%. The precision, given as the relative standard deviation, at three different concentrations (QC samples) was consistently about 3% to 10%. The limit of quantification was 2 nM.

Rapid and Sensitive Method for Determination of Cyclophosphamide in Patients Plasma Samples Utilizing Microextraction by Packed Sorbent Online with Liquid Chromatography‐Tandem Mass Spectrometry (MEPS‐LC‐MS/MS)

Abstract The aim of the present investigation was to develop a simple and fast method for the determination of cyclophosphamide in human plasma samples. Microextraction in packed syringe (MEPS) was used as an online rapid sample preparation method, followed by liquid chromatography with tandem mass spectrometry (LC‐MS‐MS) for the quantification of cyclophosphamide. The new method reduced the sample handling and the analysis time by several folds compared to liquid chromatography and UV detection. The limit of detection (LOD) was 0.005 µg/mL and the lower limit of quantification was 0.5 µg/mL. The accuracy of the quality control (QC) samples ranged from 95 to 106%. The inter‐day variation was within the range 5–9% while the intra day variation was between 1–5%. The calibration curve in plasma was constructed within the concentration range 0.5–150 µg/mL. The regression correlation coefficient (r) was ≥0.99 for all runs. The limit of detection improved by 100 time using MEPS‐LC‐MS/MS (0.005 µg/mL) compared to LLE‐LC‐UV (0.5 µg/mL). The present method was employed for the analysis of human plasma samples for more 170 patient samples. The concentrations obtained from LC‐MS‐MS were in good agreement with these obtained from LC‐UV with the ratio of 1.02±0.11. The present method is rapid, reliable, and robust and may be used for therapeutic drug monitoring of cyclophosphamide.

Microextraction in Packed Syringe/Liquid Chromatography/Electrospray Tandem Mass Spectrometry for Quantification of Acebutolol and Metoprolol in Human Plasma and Urine Samples

Abstract The aim of the present investigation was to develop a simple, fast, and sensitive method for the determination of acebutolol and metoprolol in human plasma and urine samples. The determination of acebutolol and metoprolol in plasma and urine was performed using micro extraction in packed syringe (MEPS) as a sample preparation method, online with high performance liquid chromatography and tandem mass spectrometry (LC‐MS/MS). In MEPS the sampling sorbent was 1 mg polystyrene polymer, which was inserted in a 250 µL syringe. The lower limits of quantification (LLOQ) for acebutolol and metoprolol were set to 1.0 ng/mL. The accuracy of quality control samples (QC) varied by ±10%, and precision (R.S.D.) had a deviation of 1.4–12% for plasma and urine samples. The calibration curve was obtained within the concentration range 1.0–100 ng/mL in both plasma and urine. The regression correlation coefficients (R2) for plasma and urine samples were ≥0.999 for all runs. The present method is miniaturized, fully automated, robust, and can be easily used for pharmacokinetic and pharmacodynamic studies of acebutolol and metoprolol.

High-performance liquid chromatography-tandem electrospray mass spectrometry for the determination of lidocaine and its metabolites in human plasma and urine.

A sensitive, selective and accurate high-performance liquid chromatographic-tandem mass spectrometric assay was developed and validated for the determination of lidocaine and its metabolites 2,6-dimethylaniline (2,6-xylidine), monoethylglycinexylidide and glycinexylidide in human plasma and urine. A simple sample preparation technique was used for plasma samples. The plasma samples were ultrafiltered after acidification with phosphoric acid and the ultrafiltrate was directly injected into the LC system. For urine samples, solid-phase extraction discs (C(18)) were used as sample preparation. The limit of quantification (LOQ) was improved by at least 10 times compared to the methods described in the literature. The LOQ was in the range 1.6-5 nmol/l for the studied compounds in plasma samples.

Simultaneous Determination of Busulphan in Plasma Samples by Liquid Chromatography‐electrospray Ionization Mass Spectrometry Utilizing Microextraction in Packed Syringe (MEPS) as On‐line Sample Preparation Method

Abstract Busulphan is an alkylating agent used in high doses as preparative regimen before stem cell transplantation (SCT). Busulphan has a narrow therapeutic window and under‐ or overdosing may have a fatal outcome for the patient. Therapeutic drug monitoring followed by dose adjustment is currently used to adjust the exposure to busulphan. This is an important issue to optimise and individualise high dose therapy with busulphan. However, this approach is limited to centres with experienced personal measuring busulphan plasma concentrations. An automated and easy‐to‐handle method for measurement of busulphan plasma concentrations may facilitate and wide‐spread drug monitoring approach and thus improve the outcome of the patients undergoing SCT. Microextraction in packed syringe (MEPS) in combination with liquid chromatography and electrospray ionization mass spectrometric detection (LC‐MS/LC‐MS‐MS) to quantify busulphan in human plasma samples without derivatization was developed. MEPS is a new miniaturised, solid‐phase extraction technique that can be connected on‐line to GC or LC without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100–250 µL) as a plug. The validation of the method showed that the selectivity, accuracy and precision for the method were satisfactory. This is well in line with the international criteria for the study validation. The present method has shortened extraction time considerably and the method is fully automated, which benefits therapeutic drug monitoring of busulphan in SCT.