Liquid Chromatography-isotope Dilution Mass Research Articles

High sensitivity LC-MS methods for quantitation of hydroxy- and keto-androgens.

The quantitation of androgens is necessary to diagnose and monitor the development of diseases such as prostate cancer and polycystic ovary syndrome. Androgen measurements also support the laboratory-based study of androgen metabolism in cellular and animal models. The methods described in this chapter combine chemical derivatization of hydroxy- and keto-androgens with stable isotope dilution liquid chromatography mass spectrometry (SID-LC-MS). Chemical derivatization of hydroxy-androgens by picolinic acid and keto-androgens by Girard P enhances the ionization and detection sensitivity of androgens, while chromatographic separation and [13C]-labeled internal standards add specificity that allow for simultaneous quantitation of multiple androgens. This chapter describes the materials and protocols necessary for chemical derivatization, enzymatic synthesis of internal standards, and LC-MS detection of keto- and hydroxy-androgens.

Certification of visinin-like protein-1 (VILIP-1) certified reference material by amino acid-based and sulfur-based liquid chromatography isotope dilution mass spectrometry.

As an emerging neurodegenerative disease, Alzheimer's disease (AD) has become a leading cause of dementia in older adults. Visinin-like protein-1 (VILIP-1) is an increasingly used biomarker for AD besides the widely accepted Aβ1-40, Aβ1-42, and tau. However, significant variations exist in the commercial immuno-based assays for VILIP-1 quantification, underlining the necessity to establish a traceability chain. Certified reference materials (CRMs) located at the top of the traceability chain are traceability sources for relevant matrix standard materials. In this work, VILIP-1 solution CRM with a certified value and uncertainty of 39.82±1.52 μg·g-1 was developed and certified using amino acid-based isotope dilution mass spectrometry (AA-ID-MS) and sulfur-based isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). Certified values from both strategies showed great consistency, with traceability to SI units. Moreover, the candidate VILIP-1 CRM shows excellent homogeneity and can be stable for at least 7 days at -20°C and 12 months at -70°C. The VILIP-1 CRM developed can be used in value assignment to secondary calibrators and clinical matrix CRMs, showing prospects in early diagnosis and disease monitoring for AD.

Perfluorooctanesulfonate Can Cause Negative Bias in Creatinine Measurement in Hemodialysis Patients Using Polysulfone Dialysis Membranes.

Serum creatinine is an important clinical marker for renal clearance. However, two conventional methods (Jaffe and enzymatic) are prone to interferences with organic compounds as compared to the standard method (isotope dilution–liquid chromatography–mass spectrometry) and can cause a significant negative bias. Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) are two common perfluorochemicals (PFCs) that can easily be accumulated in humans. We aimed to verify whether this bias is the result of an accumulation of PFCs. The serum creatinine values of 124 hemodialysis patients were analyzed using the three methods. We also aimed to evaluate which biochemical parameters will influence the difference between the conventional methods and the standard method. We found that a significant underestimation occurred when using the conventional methods. Albumin is an independent factor associated with negative bias, but it loses this correlation after dialysis, likely due to the removal of protein-bound uremic toxins. PFOS can cause negative bias when using the enzymatic method. Furthermore, this linear correlation is more significant in patients who used polysulfone-based dialysis membranes, possibly due to the better clearance of other uremic toxins. The serum creatinine of uremic patients can be significantly underestimated when using conventional methods. PFCs, as well the type of dialysis membrane being used, can be influencing factors.

Creatinine standardization: a key consideration in evaluating whole blood creatinine monitoring systems for CKD screening

Early detection of CKD using point of care creatinine and eGFR testing improves patient management outcomes. We undertook a field study to evaluate the use of a whole blood creatinine/eGFR device to screen a rural Nicaraguan population to determine the variability between creatinine methods and specimen types. All specimens including capillary and venous dried blood spots (DBS) were tested with an isotope dilution liquid chromatography mass spectrometry (ID-LCMS) gold standard method. This is to our knowledge the first time a capillary whole blood (POC) method has been directly compared to the gold standard IDMS method, through the novel approach of using dried blood spots. Capillary and venous whole blood specimens were obtained and tested directly with the BCMS method, and then, DBS samples were prepared. Venous plasma specimens were tested using three laboratory analyzer creatinine methods. DBS were sent to the site performing ID-LCMS. Control samples were also prepared to assess the stability of shipment and storage of DBS. The ID-LCMS method was aligned using primary and secondary standards. Sixty-six (66) patients participated in the study, and the CKD prevalence rate was 7.8%. While all creatinine methods showed a good correlation to ID-LCMS, there was a positive bias (mean absolute bias range: 0.21–0.63 mg/dL). All methods used were 100% sensitive, but specificity varied from 62.7 to 94.9% with PPV ranging from 25 to 62.5%. A correction factor was used to align the values from each method to ID-LCMS which improved the specificity of each method. This study used a unique DBS approach to align capillary whole blood creatinine to ID-LCMS. To ensure reliability of BCMS for identifying screened patients with CKD, it is important to establish IDMS traceability and alignment prior to undertaking CKD studies.

Quantitative Bioreactor Monitoring of Intracellular Bacterial Metabolites in Clostridium autoethanogenum Using Liquid Chromatography-Isotope Dilution Mass Spectrometry.

We report a liquid chromatography–isotope dilution mass spectrometry method for the simultaneous quantification of 131 intracellular bacterial metabolites of Clostridium autoethanogenum. A comprehensive mixture of uniformly 13C-labeled internal standards (U-13C IS) was biosynthesized from the closely related bacterium Clostridium pasteurianum using 4% 13C–glucose as a carbon source. The U-13C IS mixture combined with 12C authentic standards was used to validate the linearity, precision, accuracy, repeatability, limits of detection, and quantification for each metabolite. A robust-fitting algorithm was employed to reduce the weight of the outliers on the quantification data. The metabolite calibration curves were linear with R2 ≥ 0.99, limits of detection were ≤1.0 μM, limits of quantification were ≤10 μM, and precision/accuracy was within RSDs of 15% for all metabolites. The method was subsequently applied for the daily monitoring of the intracellular metabolites of C. autoethanogenum during a CO gas fermentation over 40 days as part of a study to optimize biofuel production. The concentrations of the metabolites were estimated at steady states of different pH levels using the robust-fitting mathematical approach, and we demonstrate improved accuracy of results compared to conventional regression. Metabolic pathway analysis showed that reactions of the incomplete (branched) tricarboxylic acid “cycle” were the most affected pathways associated with the pH shift in the bioreactor fermentation of C. autoethanogenum and the concomitant changes in ethanol production.

An accuracy-based proficiency testing programme with metrological reference values for the mass fraction of acesulfame potassium and sucralose in cake mix flour

This paper describes a metrological approach to evaluate the measurement capability of laboratories participating in a then Asia Pacific Laboratory Accreditation Cooperation [APLAC, now Asia Pacific Accreditation Cooperation] proficiency testing (PT) programme on mass fraction of acesulfame potassium and sucralose in cake mix flour. The programme was jointly organised by the Chemical Metrology Laboratory of the Health Sciences Authority, Singapore, and the Singapore Accreditation Council. Through the extensive APLAC network of accreditation bodies, 41 nominated laboratories from 32 economies registered, of which 40 laboratories submitted their results. Reference values of artificial sweeteners in the programme for performance evaluation were obtained from a high-accuracy liquid chromatography isotope dilution mass spectrometry (LC-IDMS) method. One of the unique features of this PT programme was the provision of pure substance certified reference materials (CRMs) of acesulfame potassium and sucralose to the participating laboratories as calibration standards to ensure comparability and metrological traceability of results. The participating laboratories’ performance, based on z-scores, were evaluated using the assigned reference value determined from IDMS. Five and one of the laboratories’ results would be incorrectly evaluated using consensus value calculated from the laboratories’ results for performance evaluation and using commercial standard as calibrant, respectively. This PT programme demonstrates the importance of the use of metrologically traceable reference values as the assigned values for appropriate evaluation of accuracy and reliable assessment. The provision of CRMs also heightened the testing laboratories’ awareness on the importance of using CRMs as calibrants for accurate measurements.

Development of an Isotope Dilution Mass Spectrometry Method for Accurate Determination of Thiamine in Diverse Food Matrices

Many foods contain thiamine as a natural or fortified nutrient. In order to realize accurate measurement of thiamine in various food matrices, analytical methods should be developed on the basis of careful examination of the matrix and the analytes in the matrix. An isotope dilution liquid chromatography–mass spectrometry (ID-LC-MS/MS) method was developed for the analysis of thiamine in various samples such as multivitamin tablets, infant formula, rice, and wheat flour. After using spiked isotope-labeled thiamine HCl (13C4-thiamine HCl) as an internal standard, samples were analyzed by LC/MS in a selected reaction monitoring mode to detect ions at m/z 265 → 122 and m/z 269 → 122 for thiamine and 13C4-thiamine, respectively. To improve the accuracy and precision of this method, the preparation procedure was optimized depending on the chemical form of thiamine present in each sample. Thiamine in multivitamin tablets, infant formula, rice, and wheat flour was analyzed after buffer extraction, acid hydrolysis, and continuous acid and enzyme hydrolyses, respectively. The instrumental analyses were also optimized for individual samples, facilitating excellent repeatability (0.16–0.81%) and reproducibility (0.1–4%). The accuracy of the developed method was verified by comparing the experimental data with the certified values of certified reference materials. Thus, optimization of the preparation procedures and ID-LC-MS/MS method allowed the accurate determination of thiamine in diverse food matrices.

Stable Isotope Labeled DNA: A New Strategy for the Quantification of Total DNA Using Liquid Chromatography-Mass Spectrometry.

Conventional DNA quantification methods require a DNA purification step that limits their reliability in estimating the original DNA amount, especially in complex matrix. To overcome this limitation, we developed a method to calibrate the variable DNA extraction efficiencies during the purification process, allowing for the accurate quantification of DNA in complex matrix. This method is based on isotope dilution-liquid chromatography-mass spectrometry using stable isotope labeled DNA (SILD) as an internal standard. Steps include spiking prepared SILD into samples, purification, enzymatic hydrolysis, and detection of DNA monomers via mass spectrometry, where the spiked SILD is expected to behave the same as the target DNA throughout the entire procedure. We show that the mean recoveries of four different DNA purification kits were dramatically improved by using the SILD internal standard, both for Escherichia coli and human genomic DNA. As standards for calibration, deoxyribonucleoside monophosphates and purified genomic DNA were tested, with genomic DNA from corresponding species found to calibrate the variable extraction efficiencies more effectively. With this successful calibration, our newly developed procedure enables International System of Units-traceable quantification of total DNA in complex matrix.

Peptides releasing study of serum apolipoprotein A-I and apolipoprotein B by ID-LC-MS

Objective To investigate the digestion kinetics of Apolipoprotein A-I and B by ID-LC-MS method for accurate quantification of proteins. Methods Methodological research. The target peptides of ApoA-I and B were determined. The ApoA-I and B from 5 human serum samples on market with levels from 0.90-2.54 g/L and 0.54-1.39 g/L separately, were measured in terms of target peptides by isotope dilution liquid chromatography mass spectrometry method. The releasing amount and rate of peptides were analyzed and plotted according to different time points. The correlation coefficient R2 was calculated among peptide releasing amount between samples. Results Most peptides reached their peaks within 4 hours. The peptides VQ, DY and VS from Apo A-I, TR and FP from Apo B were released relatively slowly. After getting to their peak stage, the ratio between TEV and SIL-TEV, AK and SIL-AK, VQ and SIL-VQ presented stable state. As for Apo A-I the correlations among peptides are high, from 0.904 to 0.999. Some peptides from Apo B show lower correlations, such as TG-SV with R2 0.543 (3 h). Conclusions Peptides from Apo A-I and Apo B present different releasing properties after trypsin digestion. Proper selection of representative peptides and enzymatic conditions can benefit accurate quantification of target proteins.(Chin J Lab Med, 2018, 41: 870-874) Key words: Apolipoprotein A-I; Apolipoproteins B; Kinetics; Chromatography, liquid; Tandem mass spectrometry; Isotope labeling

Urinary proteins: up-to-date reference methods for urinary protein analysis.

Measurement of urine albumin has been introduced in the new classification of kidney disease (KD) as a marker for detecting, monitoring and predicting KD. Currently, the measure is not standardized. The variability of results obtained with commercially available procedures is important and can lead to misclassification of patients. Analytical standardization, started in 2007, is in progress. SRM 2925 primary reference material, SRM 3666 secondary reference material and liquid chromatography isotope dilution mass spectroscopy (LC-IDMS) reference measurement procedure are being validated by the National Institute of Standards and Technology (NIST). This report presents strategies and difficulties for developing this reference system.

Provision of proficiency testing for histamine mass fraction in canned tuna to improve the capability of chemical laboratories in the Philippines

In this work, two reference materials (RMs) for mass fraction of histamine in canned tuna were developed to address the need of local testing laboratories for matrix RMs and a PT scheme for histamine. A validated method using HPLC with fluorescence detector (340 nm for excitation and 445 nm for emission) was applied with post-column derivatization for the RM’s homogeneity and stability studies. Acceptable results were obtained after statistical evaluation of homogeneity and stability according to IUPAC Harmonized Protocol and ISO Guide 35, respectively. Two PT exercises for histamine determination based on ISO/IEC 17043 were organized in 2014 and 2015 with the assigned values of 148 and 65 mg/kg, respectively. These reference values were obtained using exact matching liquid chromatography–isotope dilution mass spectrometry (LC–IDMS) with gravimetric sample preparation. The corresponding uncertainty of the reference value was obtained by ISO GUM approach where the characterization of the sample gave the highest contribution (41 %). The satisfactory z-score performance evaluated from the 12 laboratories was 50 % and 58.3 % in the first and second PT round, respectively. Most of the laboratories used AOAC Official Method 977.13 but not all conducted method validation. The 50 % decrease in unsatisfactory results in the second PT round is a significant improvement in the local laboratory performance for histamine analysis. However, the provision of more PT schemes and the increased use of CRMs in the future are still needed by these laboratories for external quality assurance and method validation to enhance the present capability.

Traceability to a primary reference measurement procedure (ID-LCMS); A key step in validating the clinical accuracy and safety of hospital blood glucose monitoring systems

ObjectiveA key step in the evaluation of the accuracy of blood glucose monitoring systems (BGMS) is using a comparator method aligned to a high order definitive reference method. We describe how we achieved traceability to an isotope dilution liquid chromatography mass spectrometry (ID-LCMS) method. We used ID-LCMS to evaluate the accuracy and specificity of two hospital BGMS used in China. MethodID-LCMS was used to verify the calibration alignment of the laboratory plasma hexokinase reference method using NIST standard reference material and clinical samples. The ID-LCMS aligned hexokinase method was used to evaluate the clinical accuracy of two BGMS in hospitalized patients. System accuracy was evaluated using Chinese consensus guidelines. BGMS accuracy was also assessed with interference factors known to be present in critically ill patients' blood. ResultsThe laboratory plasma hexokinase reference method was shown to calibrate closely with ID-LCMS. Two BGMS demonstrated good correlation with this reference method. Only one BGMS met the Chinese guidelines. The interference factors didn’t influence this BGMS but adversely affected the clinical accuracy of the other. ConclusionsWe advocate that our IDMS calibration alignment approach for ensuring the accuracy of the glucose reference method should be adopted in evaluations assessing the accuracy of blood glucose monitoring systems.

Determination and quantification of arbutin in plants using stable isotope dilution liquid chromatography–mass spectrometry

Arbutin is a very safe whitening agent for human skin. Since it is more expensive than other agents and has a challenging synthesis, novel methods to obtain this valuable agent are needed. In this study, we developed a precise and accurate method to detect and quantify arbutin using stable isotope dilution liquid chromatography–mass spectrometry (LC–MS). One challenge that needed to be overcome was the matrix effect occurring during the LC–MS analysis due to the analyte ionisation enhancement or suppression in the electrospray ionisation source by co-eluting compounds. Notably, arbutin had different matrix effects in the various sample matrices. A solution to this problem was the use of [d4]-arbutin as a stable isotope-labelled internal standard (SIL-IS), as it compensated the matrix effect of arbutin because it was affected by almost the same matrix effect. The validation of the developed method showed excellent linearity (r2 = 1.000), precision (relative standard deviation ≤ 2.5%), accuracy (recovery, 97.42–98.52%), limit of detection (0.03 μg/mL), and limit of quantification (0.1 μg/mL). Finally, the method of arbutin detection was applied to blueberry leaves to compare the precision and accuracy results obtained by performing stable isotope dilution using LC–MS and gas chromatography–mass spectrometry. The method was applied to strawberry leaves and pear peels, indicating that the SIL-IS method can be expected to find application in the arbutin analysis in other plants.

Quantification of Total Vitamin-D-Binding Protein and the Glycosylated Isoforms by Liquid Chromatography-Isotope Dilution Mass Spectrometry.

Vitamin-D-binding protein (VDBP), a transporter of 25-hydroxyvitamin D metabolites, has three common isoforms. The relationship of the isoforms and their glycosylation state with various diseases has been under recent examination. In this work, liquid chromatography coupled to isotope dilution mass spectrometry was evaluated for quantification of VDBP, the three common isoforms, and total glycosylation. Protocols using guanidine, urea, RapiGest, trifluoroethanol, or tris buffer were also evaluated for optimal tryptic digestion. Differences in peptide release were detected between purified and plasma VDBP; however, for both protein sources, ELPEHTVK, TSALSAK, and VLEPTLK concentrations were reproducible between most protocols tested. The isoform-specific peptides, LPDATPK, LPDATPTELAK, and LPEATPTELAK, were optimally released when TFE was added to plasma. The total VDBP concentration calculated from the three shared peptides resulted in 97.6% accuracy compared with the concentration from amino acid analysis. Glycosylation of VDBP was also calculated for purified protein and donor samples using the ratio of the isoform-specific peptide(s) to the total protein concentration. Glycosylation of purified VDBP was found to be 99.5-111.1% the value determined by semiquantitative analysis of the intact protein by LC-MS. This approach may be used to quantify other samples containing a mixture of isoforms and post-translational modifications.

Testicular vs adrenal sources of hydroxy-androgens in prostate cancer

Neoadjuvant androgen deprivation therapy (NADT) is one strategy for the treatment of early-stage prostate cancer; however, the long-term outcomes of NADT with radical prostatectomy including biochemical failure-free survival are not promising. One proposed mechanism is incomplete androgen ablation. In this study, we aimed to evaluate the efficiency of serum hydroxy-androgen suppression in patients with localized high-risk prostate cancer under NADT (leuprolide acetate plus abiraterone acetate and prednisone) and interrogate the primary sources of circulating hydroxy-androgens using our recently described stable isotope dilution liquid chromatography mass spectrometric method. For the first time, three androgen diols including 5-androstene-3β,17β-diol (5-adiol), 5α-androstane-3α,17β-diol (3α-adiol), 5α-androstane-3β,17β-diol (3β-adiol), the glucuronide or sulfate conjugate of 5-adiol and 3α-adiol were measured and observed to be dramatically reduced after NADT. By comparing patients that took leuprolide acetate alone vs leuprolide acetate plus abiraterone acetate and prednisone, we were able to distinguish the primary sources of these androgens and their conjugates as being of either testicular or adrenal in origin. We find that testosterone, 5α-dihydrotestosterone (DHT), 3α-adiol and 3β-adiol were predominately of testicular origin. By contrast, dehydroepiandrosterone (DHEA), epi-androsterone (epi-AST) and their conjugates, 5-adiol sulfate and glucuronide were predominately of adrenal origin. Our findings also show that NADT failed to completely suppress DHEA-sulfate levels and that two unappreciated sources of intratumoral androgens that were not suppressed by leuprolide acetate alone were 5-adiol-sulfate and epi-AST-sulfate of adrenal origin.

Evaluation of bilirubin interference and accuracy of six creatinine assays compared with isotope dilution–liquid chromatography mass spectrometry

ObjectivesThe aim of this study was to estimate bilirubin interference and accuracy of six routine methods for measuring creatinine compared with isotope dilution–liquid chromatography mass spectrometry (ID–LC/MS). Design and methodsA total of 40 clinical serum samples from 31 patients with serum total bilirubin concentration >68.4μmol/L were collected. Serum creatinine was measured using two enzymatic reagents and four Jaffe reagents as well as ID–LC/MS. Correlations between bilirubin concentration and percent difference in creatinine compared with ID–LC/MS were analyzed to investigate bilirubin interference. Bias estimations between the six reagents and ID–LC/MS were performed. Recovery tests using National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 967a were also performed. ResultsBoth the enzymatic methods showed no bilirubin interference. However, three of the four Jaffe methods demonstrated significant bilirubin concentration-dependent interference in samples with creatinine levels <53μmol/L, and two of them showed significant bilirubin interference in samples with creatinine levels ranging from 53.0 to 97.2μmol/L. Comparison of these methods with ID–LC/MS using patients' samples with elevated bilirubin revealed that the tested methods failed to achieve the bias goal at especially low levels of creatinine. In addition, recovery test using NIST SRM 967a showed that bias in one Jaffe method and two enzymatic methods did not achieve the bias goal at either low or high level of creatinine, indicating they had calibration bias. One enzymatic method failed to achieve all the bias goals in both comparison experiment and recovery test. ConclusionsIt is important to understand that both bilirubin interference and calibration traceability to ID–LC/MS should be considered to improve the accuracy of creatinine measurement.

Performance evaluation of four 25-hydroxyvitamin D assays to measure 25-hydroxyvitamin D2

ObjectivesThe ability of current immunoassays to accurately measure equimolar amounts of 25(OH)D2 and 25(OH)D3 has been recently questioned. This study determined serum 25(OH)D2, 25(OH)D3 and total serum 25(OH)D concentrations in healthy vitamin D2-supplemented subjects by isotope dilution liquid chromatography mass spectrometry (ID-LC-MS/MS); and, evaluated the ability of the Siemens, DiaSorin, Roche, and Abbott Vitamin D Total assays to monitor total serum 25(OH)D concentrations compared to an ID-LC-MS/MS method traceable to the National Institute of Standards and Technology (NIST), and that has achieved certification from the Centers for Disease Control and Prevention (CDC) Vitamin D Standardization Certification Program (VDSCP). Design and methodsTwenty (20) healthy adults, with no history of prior vitamin D supplementation were administered oral vitamin D2 (2400IU/day for 6months). Serum samples (140) from baseline and monthly blood draws were tested. ResultsAfter one month, the mean serum 25(OH)D2 concentrations rose from 0.8 to 43.6nmol/L, whereas 25(OH)D3 concentrations declined from 84.0 to 63.4nmol/L; total serum 25(OH)D concentrations rose from 86.6 to 107.0nmol/L. The overall mean bias to ID-LC-MS/MS was −7.1% for the Siemens ADVIA Centaur assay, −15.3% for the DiaSorin LIAISON assay; −8.4% for the Roche ELECSYS assay and −16.3% for the Abbott ARCHITECT assay. Correlation coefficients (r) were 0.94, 0.79, 0.74, and 0.73; the mean bias for baseline [25(OH) D3-containing] versus six-month [25(OH)D2- and 25(OH)D3-containing] samples was −13.4% and −5.7%; −3.5% and 20.3%, 9.6% and −12.1%, and 0.2% and −17.8%, respectively. ConclusionsThe bias results obtained for the Siemens ADVIA Centaur assay and Roche ELECSYS assay were slightly lower than those for the DiaSorin LIAISON assay and the Abbott ARCHITECT assay, but all 25(OH)D assays demonstrated acceptable performance.

Acute resistance exercise induces antinociception by activation of the endocannabinoid system in rats.

Resistance exercise (RE) is also known as strength training, and it is performed to increase the strength and mass of muscles, bone strength, and metabolism. RE has been increasingly prescribed for pain relief. However, the endogenous mechanisms underlying this antinociceptive effect are still largely unexplored. Thus, we investigated the involvement of the endocannabinoid system in RE-induced antinociception. Male Wistar rats were submitted to acute RE in a weight-lifting model. The nociceptive threshold was measured by a mechanical nociceptive test (paw pressure) before and after exercise. To investigate the involvement of cannabinoid receptors and endocannabinoids in RE-induced antinociception, cannabinoid receptor inverse agonists, endocannabinoid metabolizing enzyme inhibitors, and an anandamide reuptake inhibitor were injected before RE. After RE, CB1 cannabinoid receptors were quantified in rat brain tissue by Western blot and immunofluorescence. In addition, endocannabinoid plasma levels were measured by isotope dilution-liquid chromatography mass spectrometry. RE-induced antinociception was prevented by preinjection with CB1 and CB2 cannabinoid receptor inverse agonists. By contrast, preadministration of metabolizing enzyme inhibitors and the anandamide reuptake inhibitor prolonged and enhanced this effect. RE also produced an increase in the expression and activation of CB1 cannabinoid receptors in rat brain tissue and in the dorsolateral and ventrolateral periaqueductal regions and an increase in endocannabinoid plasma levels. The present study suggests that a single session of RE activates the endocannabinoid system to induce antinociception.

Development and validation of a liquid chromatography isotope dilution mass spectrometry method for the reliable quantification of alkylphenols in environmental water samples by isotope pattern deconvolution

We present here a new measurement method for the rapid extraction and accurate quantification of technical nonylphenol (NP) and 4-t-octylphenol (OP) in complex matrix water samples by UHPLC-ESI-MS/MS. The extraction of both compounds is achieved in 30min by means of hollow fiber liquid phase microextraction (HF-LPME) using 1-octanol as acceptor phase, which provides an enrichment (preconcentration) factor of 800. On the other hand we have developed a quantification method based on isotope dilution mass spectrometry (IDMS) and singly 13C1-labeled compounds. To this end the minimal labeled 13C1-4-(3,6-dimethyl-3-heptyl)-phenol and 13C1-t-octylphenol isomers were synthesized, which coelute with the natural compounds and allows the compensation of the matrix effect. The quantification was carried out by using isotope pattern deconvolution (IPD), which permits to obtain the concentration of both compounds without the need to build any calibration graph, reducing the total analysis time. The combination of both extraction and determination techniques have allowed to validate for the first time a HF-LPME methodology at the required levels by legislation achieving limits of quantification of 0.1ngmL−1 and recoveries within 97–109%. Due to the low cost of HF-LPME and total time consumption, this methodology is ready for implementation in routine analytical laboratories.

Overcoming matrix effects in electrospray: Quantitation of β-agonists in complex matrices by isotope dilution liquid chromatography–mass spectrometry using singly 13C-labeled analogues

In this work, the implementation of isotope dilution mass spectrometry (IDMS) using minimal labeling and isotope pattern deconvolution (IPD) is evaluated as a strategy for the minimization of matrix effects during trace determination of β2-agonists in complex matrices by liquid chromatography electrospray ionization mass spectrometry (LC–ESI-MS). First, the parameters affecting the measurement of isotopic composition of organic compounds by liquid chromatography electrospray ionization high resolution mass spectrometry with a time-of-flight analyzer were evaluated using as a case of study three different β2-agonists: clenbuterol, clenproperol and brombuterol. Then, a calibration graph-free IDMS methodology was evaluated in order to overcome matrix effects in LC–ESI-MS in complex samples. In this procedure singly 13C-labeled analogues of clenbuterol, clenproperol and brombuterol were employed in combination with IPD. Using this approach accurate and precise results were obtained in the simultaneous quantification of β2-agonists in human urine and bovine liver, even at the sub ngg−1 and particularly in spite of the previously reported matrix effects. Recovery rates in the range of 97–114% in fortified human urine and from 95% to 111% in fortified bovine liver were obtained with RSD (%) of independent recovery experiments always lower than 6%. These results demonstrate that the proposed methodology based on the use of 13C1-labeled standards and IPD is a reliable approach for accurate LC–MS quantitation of small molecules and compatible with full-scan high-resolution mass spectrometry.