Determination of N-acetyl retigabine in dog plasma by LC/MS/MS following off-line microElution 96-well solid phase extraction

A high throughput off-line microElution 96-well solid phase extraction (SPE) followed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for the determination of N-acetyl retigabine in dog plasma has been developed and validated. The method involves the use of microElution 96-well SPE for the simultaneous extraction of N-acetyl retigabine and rapid removal of its N-glucuronide metabolite that has shown to be problematic due to its instability using other clean-up methods. The microElution SPE technology eliminates the need for post-extraction solvent evaporation and greatly reduces sample preparation time consequently improving assay efficiency.     

Systematic and comprehensive strategy for reducing matrix effects in LC/MS/MS analyses

A systematic, comprehensive strategy that optimizes sample preparation and chromatography to minimize matrix effects in bioanalytical LC/MS/MS assays was developed. Comparisons were made among several sample preparation methods, including protein precipitation (PPT), liquid-liquid extraction (LLE), pure cation exchange solid-phase extraction (SPE), reversed-phase SPE and mixed-mode SPE. The influence of mobile phase pH and gradient duration on the selectivity and sensitivity for both matrix components and basic analytes was investigated. Matrix effects and overall sensitivity and resolution between UPLC technology and HPLC were compared. The amount of specific matrix components, or class of matrix components, was measured in the sample preparation extracts by LC/MS/MS with electrospray ionization (ESI) using both precursor ion scanning mode and multiple reaction monitoring (MRM). PPT is the least effective sample preparation technique, often resulting in significant matrix effects due to the presence of many residual matrix components. Reversed-phase and pure cation exchange SPE methods resulted in cleaner extracts and reduced matrix effects compared to PPT. The cleanest extracts, however, were produced with polymeric mixed-mode SPE (both reversed-phase and ion exchange retention mechanisms). These mixed-mode sorbents dramatically reduced the levels of residual matrix components from biological samples, leading to significant reduction in matrix effects. LLE also provided clean final extracts. However, analyte recovery, particularly for polar analytes, was very low. Mobile phase pH was manipulated to alter the retention of basic compounds relative to phospholipids, whose retention tends to be relatively independent of pH. In addition to the expected resolution, speed and sensitivity benefits of UPLC technology, a paired t-test demonstrated a statistically significant improvement with respect to matrix effects when this technology was chosen over traditional HPLC. The combination of polymeric mixed-mode SPE, the appropriate mobile phase pH and UPLC technology provides significant advantages for reducing matrix effects resulting from plasma matrix components and in improving the ruggedness and sensitivity of bioanalytical methods.     

Matrix solid‐phase dispersion with sand in chromatographic analysis of essential oils in herbs

Introduction – Matrix solid‐phase dispersion (MSPD) is a very simple, cheap and relatively quick sample preparation procedure which involves simultaneous disruption and extraction of various solid and semi‐solid samples due to the direct mechanical blending of the sample with a SPE sorbent, mainly C₁₈. Little is known about MSPD application as a sample preparation method for the analysis of essential oil components in herbs. Objective – To evaluate if C₁₈ sorbent, commonly used in MSPD process, can be substituted with sand in the procedure of essential oil analysis. Methodology – Essential oil extracts were obtained from mint, sage, chamomile, marjoram, savory and oregano using MSPD with C₁₈ sorbent or sand, pressurised liquid extraction and steam distillation. Their qualitative and quantitative compositions ware established by GC‐MS and GC‐FID. Results – The results prove that C₁₈ sorbent can be substituted with sand in the procedure of essential oil analysis in herbs. The recoveries of essential oil components estimated using MSPD/sand are almost equal to those using pressurised liquid extraction. Conclusion – The results presented in the paper reveal that MSPD with sand is suitable for the isolation of essential oil components from herbs. Its extraction efficiency is equivalent to pressurised liquid extraction, recognised as one of the most efficient extraction methods. The cost of MSPD procedure for essential oil analysis can be significantly diminished by substituting C₁₈ with sand. Copyright © 2010 John Wiley & Sons, Ltd.     

The analysis of pyrrolizidine alkaloids in Jacobaea vulgaris; a comparison of extraction and detection methods

Introduction – Pyrrolizidine alkaloids (PAs) serve an important function in plant defence. Objective – To compare different extraction methods and detection techniques, namely gas chromatography with nitrogen phosphorus detection (GC‐NPD) and liquid chromatography tandem mass spectrometry (LC‐MS/MS) with quadrupole analysers for analysing PAs in Jacobaea vulgaris. Methodology – Both formic acid and sulfuric acid were tested for PA extraction from dry plant material. For GC‐NPD, reduction is required to transform PA N‐oxides into tertiary amines. Zinc and sodium metabisulfite were compared as reducing agents. Results – The lowest PA concentration measured with GC‐NPD was approximately 0.03 mg/g and with LC‐MS/MS 0.002 mg/g. The detection of major PAs by both techniques was comparable but a number of minor PAs were not detected by GC‐NPD. With the LC‐MS/MS procedure higher concentrations were found in plant extracts, indicating that losses may have occurred during the sample preparation for the GC‐NPD method. Zinc proved a more effective reducing agent than sodium metabisulfite. The sample preparation for LC‐MS/MS analysis using formic acid extraction without any reduction and purification steps is far less complex and less time consuming compared to GC‐NPD analysis with sulfuric acid extraction and PA N‐oxide reduction with zinc and purification. Conclusions – In terms of sensitivity and discrimination, formic acid extraction in combination with LC‐MS/MS detection is the method of choice for analysing PAs (both free and N‐oxides forms) in plant material. Copyright © 2009 John Wiley & Sons, Ltd.     

An accurate quantitative LC/ESI-MS/MS method for sirolimus in human whole blood

Sirolimus is a widely used immunosuppressant that requires therapeutic drug monitoring (TDM). We optimized a preanalytical procedure that allows for the accurate quantiation of sirolimus in whole blood by LC/ESI-MS/MS with minimal matrix effects. Sirolimus is highly lipophilic, and solvents containing greater than 50% methanol were required to maintain sirolimus recovery. The final pretreatment procedure developed consists of a zinc sulfate protein precipitation, an extraction using octadecyl silyl-silica gel for eliminating water-soluble and hydrophilic compounds, and HybridSPE cartridge treatment to eliminate phospholipids. Using this procedure prior to LC/ESI-MS/MS led to the accurate and reproducible quantitation of sirolimus in human whole blood. The linear range of detection was 0.5-50 ng/mL, a range appropriate for TDM, and the method demonstrated good repeatability and intermediate precision within this quantitative range. In order to investigate the quantitative performance of this method, we compared it to two commercially available sirolimus immunoassays and our previously reported LC/ESI-MS/MS method. The immunoassays gave consistently greater values for the sirolimus concentration, and this may be related to antibody cross-reactivity with sirolimus metabolites and/or other matrix effects. Although our procedure is too long to support real-time TDM for outpatients, it can serve as reference method to assess the performance of other analytical methods that are currently available or may be developed in the future.     

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.     

Control of matrix effects in the analysis of urinary F2-isoprostanes using novel multidimensional solid-phase extraction and LC-MS/MS

F(2)-isoprostanes (F(2)-iPs), established markers of oxidative stress, exist as four sets of regioisomers. Simultaneous and specific determination of F(2)-iPs can be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed novel methods for urine sample preparation and HPLC to control matrix-related ion suppression effects in the LC-MS/MS analysis of F(2)-iPs. A selective solid-phase extraction (SPE) wash protocol was developed with an Oasis HLB (hydrophilic-lipophilic balance) SPE cartridge using an elution profile of [(3)H]8-iso-prostaglandin (PG)F(2alpha) (iPF(2alpha)-III) when the methanol concentration was increased under acidic, neutral, and base wash conditions. A multidimensional (MD)-SPE method that incorporated size exclusion chromatography [corrected] reverse-phase chromatography, and normal-phase chromatography was developed using an Oasis HLB SPE cartridge and an HLB microElution SPE plate. Average extraction recoveries of the deuterated internal standards of iPF(2alpha)-III and iPF(2alpha)-VI were 62 +/- 8% and 60 +/- 10%. A buffer-free HPLC method for the separation of F(2)-iP isomers was developed on base-deactivated C8 columns. Average matrix effects for iPF(2alpha)-III and iPF(2alpha)-VI were 95 +/- 6% and 103 +/- 5%. The clean extraction of urine F(2)-iPs using MD-SPE and the separation of F(2)-iP isomers using a novel HPLC method did not cause apparent ion suppression in the analysis of iPF(2alpha)-III and iPF(2alpha)-VI using LC-MS/MS. These findings should be useful for establishing a routine LC-MS/MS method for the analysis of F(2)-iPs.     

Quantitative secondary ion monitoring gas chromatography/mass spectrometry of diethylstilbestrol in bovine liver

A procedure is described for the extraction of diethylstilbestrol (DES) from animal tissue for quantitative capillary gas chromatography/mass spectrometry (GC/MS). The procedure is based upon use of a strong anion exchange polystyrene divinylbenzene resin for sample purification. The recovery of DES from the resin clean up was 88% in the high parts per trillion (ppt) range. Criteria for identification of DES using selected ion monitoring (SIM) GC/MS are presented. Liquid chromatography/mass spectrometry (LC/MS) was used to investigate altered DES cis/trans ratios observed in biological extracts.     

Rapid screening of lignans from Phyllanthus myrtifolius and stilbenoids from Syagrus romanzoffiana by HPLC-SPE-NMR

Introduction – Application of on‐line solid‐phase extraction (SPE) as an interface between HPLC and NMR has gained great improvement in solving sensitivity problems and signal interferences by the eluents. Objective – Rapid analysis and characterisation by HPLC‐SPE‐NMR and LC/MS of the arylnaphthalene‐type lignans present in Phyllanthus myrtifolius and the minor stilbenoids present in the polyphenol‐rich fraction from the ethanol extract of the seeds of Syagrus romanzoffiana. Methodology – Pretreatment of fractions by liquid–liquid partitioning, followed by Sephadex LH‐20 fractionation, was found very useful to facilitate the focusing and analysis of the polyphenolic fraction. HPLC‐DAD‐SPE‐NMR (400 MHz and 600 MHz) analysis was carried out using an Agilent 1100 liquid chromatography, followed by a Prospekt 2 automated solid‐phase extraction unit, containing 96 HySphere‐Resin GP cartridges (10 × 2 mm, 10–12 µm), which was connected to a 120 or 60 µL LC probe. Results – Seven arylnaphthalene‐type lignans from the chloroform‐soluble fraction of P. myrtifolius and nine stilbenoids from a polyphenol‐rich butanol‐soluble fraction of the seeds of S. romanzoffiana were characterised. Conclusion – HPLC‐SPE‐NMR associated with HR‐ESI/MS, which consumed only analytical amounts of partially purified mixtures, was demonstrated to be a good tool for rapid screening of both known and new natural products. Copyright © 2011 John Wiley & Sons, Ltd.     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

Development of an HPLC–MS procedure for the quantification of N-acetyl-S-(n-propyl)-l-cysteine,the major urinary metabolite of 1-bromopropane in human urine

An analytical procedure was developed for the detection and quantification of N-acetyl-S-(n-propyl)-l-cysteine (n-propylmercapturic acid, AcPrCys), a metabolite and biomarker for exposure to 1-bromopropane (1-BP). 1-BP is used as an industrial solvent and exposure is a health concern for industrial workers due to its toxicity. It has been associated with neurological disorders in both animals and humans. Urine sample preparation for the determination of AcPrCys consisted of solid phase extraction (SPE). Urine samples on preconditioned SPE (C18) columns were washed with 40% methanol/60% water solution prior to elution with acetone. Quantification was by means of a liquid chromatograph (LC) equipped with a mass spectrometer (MS) using an Aqua 3 μm C18 300A column and [d₇]-AcPrCys was used as internal standard. Electrospray ionization (ESI) was used with the MS operated in the negative ion mode and selected ion monitoring (SIM) at m/z 204 for AcPrCys and m/z 211 for [d₇]-AcPrCys. Demonstrated recovery of urine samples fortified at multiple levels (0.625–10 μg/ml) varied between 96 and 103% of theory with relative standard deviations (RSD) of 6.4% or less. The limit of detection (LOD) for the procedure was approximately 0.01 μg/ml AcPrCys in urine. These data will be discussed as well as other factors of the development of this test procedure.     

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two‐dimensional gel electrophoresis

Introduction – A variety of sample preparation protocols for plant proteomic analysis using two‐dimensional gel electrophoresis (2‐DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. Objective – This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2‐DE. Methodology – Four sample preparation methods were tested: (1) phenol extraction and methanol–ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid–acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS–PAGE (1‐DE) and 2‐DE. Fifteen selected protein spots were trypsinised and analysed by matrix‐assisted laser desorption/ionisation time‐of‐flight tandem mass spectrometry (MALDI‐TOF‐MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Results – Methods number 3 and 4 resulted in large quantities of protein with good 1‐DE separation and were chosen for 2‐DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. Conclusion – The described sample preparation method allows the proteomic analysis of papaya leaves by 2‐DE and mass spectrometry (MALDI‐TOF‐MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

Synthesis of a molecularly imprinted polymer for the solid‐phase extraction of betulin and betulinic acid from plane bark

Introduction – Plant extracts are usually complex mixtures of various polarity compounds and their study often includes a purification step, such as solid‐phase extraction (SPE), to isolate interest compounds prior analytical investigations. Molecularly imprinted polymers (MIPs) are a new promising type of SPE material which offer tailor‐made selectivity for the extraction of trace active components in complex matrices. Numerous specific cavities that are sterically and chemically complementary of the target molecules, are formed in imprinted polymers. A molecularly imprinted polymer (MIP) was synthesised in order to trap a specific class of triterpene, including betulin and betulinic acid from a methanolic extract of plane bark. Methodology – Imprinted polymers were synthesised by thermal polymerisation of betulin as template, methacrylic acid (MAA) or acrylamide (AA) as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and chloroform as porogen. Afterwards, MAA‐ and AA‐MIPs were compared with their non‐imprinted polymers (NIPs) in order to assess the selectivity vs betulin and its derivatives. Recovered triterpenes were analysed by HPLC during MIP‐SPE protocol. Results – After SPE optimisation, the MAA‐imprinted polymer exhibited highest selectivity and recovery (better than 70%) for betulin and best affinity for its structural analogues. Thus, a selective washing step (chloroform, acetonitrile) removed unwanted matrix compounds (fatty acids) from the SPE cartridge. The elution solvent was methanol. Finally, the MAA‐MIP was applied to fractionate a plane bark methanolic extract containing betulin and betulinic acid. Conclusion – This study demonstrated the possibility of direct extraction of betulin and its structural analogues from plant extracts by MIP technology. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitation of binding, recovery and desalting efficiency of peptides and proteins in solid phase extraction micropipette tips

Micropipette-tip solid phase extraction (SPE) systems are common in proteomic analyses for desalting and concentrating samples for mass spectrometry, removing interferences, and increasing sensitivity. These systems are inexpensive, disposable, and highly efficient. Here, we show micropipette-tip solid phase extraction is a direct sample preparation method for (14)C-accelerator mass spectrometry (AMS), removing salts or reagent from labeled macromolecules. We compared loading, recovery and desalting efficiency in commercially available SPE micro-tips using (14)C-labeled peptides and proteins, AMS, and alpha spectrometry ion energy loss quantitation. The polypropylene in the tips was nearly (14)C-free and simultaneously provided low-background carrier for AMS. The silica material did not interfere with the analysis. Alpha spectrometry provided an absolute measurement of desalting efficiency.     

Multi-residue extraction–purification procedure for corticosteroids in biological samples for efficient control of their misuse in livestock production

A fast and efficient multi-residue extraction–purification procedure was developed for 12 corticosteroids in biological matrices (hair, urine and meat), in order to control their illegal use as growth promoters in cattle. Detection and identification of the analytes were achieved using a previously described LC–MS–MS method based on negative electrospray ionisation and a triple quadrupole analyser. The presented procedures included acid (hair) or enzymatic (urine and meat) hydrolysis, C₁₈ reversed-phase SPE, Na₂CO₃ liquid–liquid clean-up and SiOH normal-phase SPE. The detection limits of the developed methods were between 2.9 and 9.3 pg/mg (ppb) for hair samples and in the 40 – 70 pg/g (ppt) range for the urine or meat samples. The acid hydrolysis used for corticosteroid extraction in hair was optimised using an experimental design and response surface methodology. Achieved performances were linked to a physico–chemical approach based on the corticosteroids specific C₁₇ side-chain. This original multi-residue and multi-matrices analytical methodology will be used for further metabolism studies.     

Minimizing matrix effects in the development of a method for the determination of salmeterol in human plasma by LC/MS/MS at low pg/mL concentration levels

Salmeterol is an inhaled bronchodilator drug used for treatment of asthma. Its concentrations in plasma are very low or undetectable by previously developed methods. The present paper describes a method for analysis of salmeterol in human plasma with 2.5 pg/mL lower limit of quantitation. Despite the basic character of the drug the method uses mixed mode anion-exchange solid phase extraction for sample preparation combined with a column switching approach to minimize matrix effects. Samples are separated and detected by LC/MS/MS. The method is easy to use, only requires 0.5 mL of plasma and was validated for use in bioanalytical applications. The method does not suffer from interference from co-administered fluticasone propionate.     

Superheated water extraction of essential oils from Cinnamomum zeylanicum (L.)

Introduction – Superheated water extraction (SHWE) potentially provides an environmentally friendly and clean extraction technique which uses a minimum or no organic solvent. The scope and limitations of the technique have still to be fully explored. Objective – To investigate the application of SHWE to cinnamon (Cinnamomum zeylanicum L.) bark and leaves as typical plant materials to determine if this extraction method can yield a higher quality oil. Methodology – Samples of cinnamon bark or leaves were extracted at 200°C with water under pressure. The essential oils were obtained from the aqueous solution using a solid phase extraction cartridge and were then examined by GC‐MS. Results – Using superheated water extraction, cinnamon bark oil with over 80% cinnamaldehyde and cinnamon leaf oil containing up to 98% eugenol were obtained. Alternative solvent extraction methods were also studied but led to emulsion formation apparently because of the presence of cellulose breakdown products. Conclusion – Superheated water extraction offers a cheap, environmentally friendly technique with a shorter extraction time than hydrodistillation and yielded a higher quality oil with a higher proportion of eugenol than hydrodistillation. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of tetracycline residues in royal jelly by liquid chromatography-tandem mass spectrometry

A confirmatory method coupling liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed to determine the concentration of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC), which make up the tetracycline (TC) groups present in royal jelly. Sample preparation included deproteination, control of pH, extraction and clean-up on a solid-phase extraction (SPE) cartridge. The analyses were achieved by LC/MS/MS in selected reaction monitoring mode (SRM). The overall recovery of fortified royal jelly at the levels of 5.0, 10.0 and 40.0 microg/kg ranged from 62% to 115%, and the coefficients of variation ranged from 3.4% to 16.3% (n=6). The detection limits for TCs were under 1.0 microg/kg. The transformation between the TCs and its epimers (EpiTCs) was studied in standard solution and during the sample preparation process. This method can be used for the detection of tetracycline residues in royal jelly.