Seven-point Calibration Curve Research Articles

Quantitative Opiate, Opioid, and Benzodiazepine Testing by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Fatalities due to opioids and other controlled substances continue to increase year over year and maintain an epidemic level nationally. CDC data indicates that drug overdoses killed more than 100,000 Americans in 2021 with synthetic opioids being the main driver of the national crisis (Wide-ranging online data for epidemiologic research (WONDER). Multiple Cause of Death 1999-2020. Centers for Disease Control and Prevention, National Center on Health Statistics. http://wonder.cdc.gov . Accessed 28 Feb 2023, 2021). Response to this crisis includes the development of laboratory testing to support the evolving drug trends. Urine drug testing provides clinicians with objective information to assist in making treatment decisions by identifying the presence of potential drugs of abuse and assessing patient compliance to a controlled substance prescription regimen. In this chapter, we describe a rapid LC-MS/MS method for the simultaneous quantitation of 21 opiate, opioid, and benzodiazepine drugs in less than 4min. The method requires 100uL of urine and includes enzymatic hydrolysis of glucuronide conjugates using β-glucuronidase to provide total drug measurement. Quantitation is accomplished using multiple reaction monitoring (MRM), a seven-point calibration curve, and deuterated internal standards. This method provides a robust and reliable means to measure commonly prescribed opioid (including natural, semi-synthetic, and synthetic) and benzodiazepine drugs in urine.

Targeted Metabolomics Approach Identifies Alterations in the Plasma Metabolome of Multiple Myeloma Patients with and without Extramedullary Spread

Introduction: Metabolomics refers to the identification and quantitation of metabolites in cells, tissues and biofluids. The metabolome (complete set of metabolites in a biological sample) reflects the biochemical events occurring in an organism at a given time, thus providing a valuable source to analyse metabolic changes in a variety of diseases, including cancer. Metabolic profiling of blood cancers represents a useful tool for the detection of novel biomarkers and therapeutic targets. Multiple myeloma (MM) accounts for less than 2% of all new cancer cases in the United States. The rare and aggressive sub-entity of MM, extramedullary multiple myeloma (EMM), develops when malignant plasma cells escape the bone marrow microenvironment and colonize distal tissues or organs. The prognosis of EMM is poor, and the incidence of EMM increases at disease progression, occurring in up to 30% of relapsed MM patients. The pathogenic mechanisms of EMM are poorly understood with no targeted therapies currently available to strategize treatment regimens. Here, we use a targeted metabolomics approach to identify metabolic changes in the plasma of MM patients with and without extramedullary spread. Methods: Targeted metabolomic analysis of age and gender-matched medullary MM (n=8) and EMM (n=9) blood plasma samples was performed using the MxP® Quant 500 kit (Biocrates Life Sciences AG, Innsbruck, Austria) with a SCIEX QTRAP 6500plus mass spectrometer. The MxP® Quant 500 kit is capable of quantifying more than 600 metabolites from 26 compound classes. Quality control (QC) samples were employed to monitor the performance of the analysis with metabolite concentration in each sample normalised based on these QC samples. Isotopically labelled internal standards and seven-point calibration curves were used in the quantitation of amino acids and biogenic amines. Semi-quantitative analysis of other metabolites was performed using internal standards. Data quality was evaluated by checking the accuracy and reproducibility of QC samples. Metabolites were included only when the concentrations of the metabolites were above the limit of detection (LOD) in >75% of plasma samples. Data was imported into MetaboAnalyst 5.0 for further analysis. Feature filtering was performed based on relative standard deviation (RSD) and the resulting data was autoscaled. Metabolites of interest were identified based on p-value < 0.01 and fold-change > 1.5 between experimental groups. Supervised statistical approaches were used to further interrogate the data. Results: Using a targeted metabolomic technique, we compared the metabolic profile of MM and EMM patient plasma. Univariate analysis using a t-test identified 5 metabolites of interest; HexCer(d18:1/20:0), TG(16:0_34:2), TG(22:4_32:0), TG(18:2_32:0), and Taurine (Figure 1(A)). The supervised clustering technique orthogonal projection to latent structure discriminant analysis (OPLS-DA) was used to determine separation between the two groups (MM and EMM). OPLS-DA scores plot illustrated a distinct separation between MM patients with extramedullary spread (red dots) compared to those without extramedullary spread (green dots) (Figure 1(B)). A permutation test (n=1000) was performed to ensure there was no overfitting of the data. Permutation analysis results (Q2 = 0.478, p = 0.023; R2Y = 0.978, p = 0.033) demonstrated the model was of good predictive quality. Discriminatory variables responsible for the group separation were identified using the OPLS-DA variable importance in projection (VIP) score to identify metabolites with a score greater than 1.5. HexCer(d18:1/20:0), TG(16:0_34:2), TG(22:4_32:0), TG(18:2_32:0), and Taurine had VIP scores of 2.5, 2.3, 2.1, 2.2 and 2.4, respectively. The diagnostic potential of these metabolites as EMM biomarkers was evaluated by receiver operating characteristic (ROC) curve analysis. All 5 metabolites demonstrated high diagnostic potential with area under the curve (AUC) values greater than 0.84. Conclusion: Investigating disease-associated metabolomes presents an opportunity to identify dysregulated metabolic processes and novel biomarkers. This pilot targeted metabolomic analysis of EMM plasma samples reveals metabolites of interest for further analysis and contributes to our understanding of EMM pathophysiology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Validated UPLC-MS/MS method for the analysis of vitamin B6 pyridoxal 5́-phosphate, pyridoxal, pyridoxine, pyridoxamine, and pyridoxic acid in human cerebrospinal fluid

Vitamin B6 and its metabolites play a crucial role in the development and interaction of brain metabolism. Following diagnostic improvements additional inherited disorders in vitamin B6 metabolism have been identified, most of them leading to a severe epileptic disorder accompanied by progressive neurological deficits including intellectual disability and microcephaly. Since early treatment can improve the outcome, fast and reliable detection of metabolic biomarkers is important. Therefore, the analysis of vitamin B6 metabolites has become increasingly important, but is, however, still challenging and limited to a few specialized laboratories.Until today, vitamin B6 metabolites are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using trichloroacetic acid for protein precipitation. In this work, we present the development and validation of a new, accurate and reliable method for analysis and quantification of the vitamin B6 vitamers pyridoxal 5́-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and pyridoxic acid (PA) in human CSF samples using acetonitrile for protein precipitation. The method is based on ultra-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (UPLC-ESI-MS/MS). The calibration was performed in surrogate matrix Ringer solution and metabolites were quantified by their corresponding isotopically labelled internal standards. A protein precipitation by acetonitrile was applied greatly improving chromatographic separation of the metabolites in a 4.7 min chromatographic run. The method was validated following the European Medical Agency (EMA) and Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The metabolites were quantified from 5 to 200 nmol/L with a seven-point calibration curve and minimum coefficient of regression of 0.99. The validation was performed with quality control samples at four concentration levels with surrogate matrix ringer solution and pooled CSF material. Within- and inter-day accuracy and precision in Ringer solution were within 85.4 % (PLP) and 114.5 % (PM) and from 2.6 % (PA) to 16.5 % (PLP). Within- and inter-day accuracy and precision in pooled CSF material were within 90.5 % (PN) and 120.1 % (PL) and from 1.7 % (PA) to 19.0 % (PM). The method was tested by measuring of 158 CSF samples to determine reference ranges. The B6 vitamers PLP and PL were determined in all CSF samples above 5 nmol/L while PN, PM and PA showed concentrations below or near LOQ. Probable supplementation of PLP was detected in eight CSF samples, which revealed high concentrations of PM, PN, PL, or PA, whereas PLP was in the reference range or slightly elevated. The method is suitable for the application within a routine diagnostic laboratory.

Expansion of the EZSTATSG1 Microsoft Excel Tool for ANSI/ASB Standard 036 Method Validation by the Addition of Weighted Quadratic External Calibration Models Utilizing Five-, Six- or Seven-Point Curves.

The first generation of this Microsoft (MS) Excel (Redmond, WA, USA) tool for method validation, EZSTATSG1, was designed for methods utilizing only linear calibration curves requiring seven calibration levels, and quadratic calibration models were not supported. This significantly improved version, EZSTATSG2, includes all of the features of the original template such as weighted linear calibration models, bias and precision data, dilution integrity and ion suppression. New features in this version include flexible five-, six- or seven-point calibration curves; six weighted quadratic calibration models; standardized residuals by use of frequency plots overlaid with the normal distribution function along with five-number summary data and processed sample stability. The implementation of Visual Basic for Applications in Excel UserForms prevents accidental alteration of existing formulas and also ensures that pertinent cells are relocked every time a file is reopened. The quadratic models feature the 95% confidence intervals for checking the significance of the second-order term and are fully characterized by providing the equations for the axis of symmetry, directrix and coordinates for vertex and focus. Example data of α-hydroxymidazolam demonstrate that the quadratic calibration curves fit the data more adequately than the linear models for this method. This second-generation tool summarizes all of the validation parameters of a method for both linear and quadratic calibration models. Models with the lower average sum of relative errors and higher R2 values are color-coded green, indicating the likelihood of a better fitting model. Like the EZSTATSG1, the redesigned EZSTATSG2.xltm MS Excel self-actuating validation tool and a completed PDF example are available to the scientific community for download as supplementary data. Updates to the template can be found at www.EZSTATS4validation.com.

Electrospun NiFe layered double hydroxide/Nylon 6 composite nanofibers as a sorbent for micro solid phase extraction by packed sorbent of non-steroidal anti-inflammatory drugs in human blood

In the present work, NiFe layered double hydroxide (LDH)/Nylon 6 composite nanofibers were prepared by electrospinning method and used as a new sorbent for the extraction and measurement of non-steroidal anti-inflammatory drugs (naproxen, mefenamic acid, and diclofenac) in whole blood samples. The method is based on micro solid phase extraction (µSPE) by packed sorbent followed by HPLC-UV analysis. Effective parameters on the extraction efficiency were optimized using a central composite design (CCD). In order to characterize the sorbent, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), field emission scanning electron microscopy (FESEM), energy dispersive X-ray analysis (EDX) and elemental mapping were applied. The method was fully validated based on linearity, limits of detection (LOD) and quantification (LOQ), precision, and recovery. Under the optimal conditions, LOD values were found to be 25 ng mL−1 for naproxen and diclofenac and 15 ng mL−1 for mefenamic acid. A seven-point calibration curve was obtained in the range of 75–2000 ng mL−1 for naproxen and diclofenac and 50–2000 for mefenamic acid. The method showed good linearity with coefficients of determination, r2> 0.9962, for the three drugs. In the entire analytical range, the relative standard deviations (RSD%) were less than 8.1%. Finally, the efficiency of the method was investigated for the analysis of the target analytes in human blood samples, and the recoveries were obtained in the range of 90.7–109.8%.

An Ultra-High-Pressure Liquid Chromatographic Tandem Mass Spectrometry Method for the Analysis of Benzoyl Ester Derivatized Glycols and Glycerol.

Presented is an ultra-high-pressure liquid chromatographic tandem mass spectrometry (UPLC-MS/MS) method developed for the detection of propylene glycol, glycerol, ethylene glycol and diethylene glycol using isotopically labeled standards in urine as part of ongoing studies to evaluate whether urinary propylene glycol and/or vegetable glycerin concentration are indicators of recent use. Propylene glycol and vegetable glycerol are found in many products that are consumed and used including electronic cigarettes (e-cigarettes). E-cigarettes are battery-powered devices used as an alternative to traditional cigarettes. The liquid formulations aerosolized in these devices largely consist of propylene glycol and/or vegetable glycerol. Published reports regarding the ratio of propylene glycol to glycerol content in these formulations ranged from 50:50 to 100 percent of either. For the analysis of urine specimens from both users and non-users of e-cigarettes, calibrators, controls and specimens were derivatized using benzoyl chloride prior to analysis. They were analyzed using a Waters AcQuity Xevo TQ-S Micro UPLC-MS/MS. Chromatographic separation was performed on an AcQuity UPLC BEH C18 1.7um, 2.1×50mm, column using a 20mM ammonium formate in water and 20mM ammonium formate in methanol as the mobile phase. The method was validated using SWGTOX guidelines for linearity, precision and accuracy, stability, carryover and limit of detection. The linear range was determined using a seven-point calibration curve ranging between 0.5 and 100mcg/mL. The bias for all validation controls was determined to be ±20% of the expected concentrations with CVs of <15%. A total of 124 urine specimens analyzed collected with 50 specimens collected from self-reported non-smokers (cigarettes/e-cigarettes) confirmed cotinine free using the DRI® Cotinine Assay (Thermo Scientific, Waltham, MA) and 74 specimens collected before and after 12hours self-reported e-cigarettes abstinence e-cigarette users. Propylene glycol and glycerol were determined to have concentration ranges of "none detected" to 1470 and "none detected" to 2950mcg/mL, respectively.

Therapeutic Drug Monitoring of Lacosamide by LC-MS/MS.

High-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) has become a primary analytical methodology in therapeutic drug monitoring of antiepileptic drugs (AEDs). To demonstrate the utility of LC-MS/MS in measuring drug concentrations in serum or plasma, analysis of lacosamide (Vimpat™) is discussed in this chapter. Lacosamide is an example of the newer-generation AEDs. The drug is extracted by protein precipitation and dilution of the serum specimen. A small volume of the extracted specimen is injected into a reversed-phase chromatography column, and lacosamide is identified by positive electrospray ionization (ESI) mass spectrometry in the multiple reaction monitoring (MRM) mode, which provides selectivity for quantitative analysis. A deuterated internal standard is used to correct for any loss of analyte during the process of extraction and analysis. A seven-point calibration curve and two levels of quality controls are included in each batch.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF ASPIRIN, ROSUVASTATIN, CLOPIDOGREL IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: To develop a novel, accurate, precise and linear reverse phase high-performance liquid chromatography (RP-HPLC) and Stability Indicating Assay Method (SIAMs) for simultaneous, qualitative and quantitative estimation of aspirin, rosuvastatin and clopidogrel in bulk and pharmaceutical dosage form as per International Conference on Harmonization (ICH) guidelines.Method: In the present work, good chromatographic separation was achieved by isocratic method using a BISCOF HPLC C18 column (250 mm ×4.6, 5 µm) and a mobile phase consisting of water at pH 2.51 with 0.1 % (v/v) orthophosphoric acid (OPA): acetonitrile in the ratio 50:50, at a flow rate of 1 ml/min. The effluents obtained were monitored at 237 nm with the UV-visible detector.Results: The retention time of aspirin, rosuvastatin, and clopidogrel was found to be 4.3 min, 7.6 min and 16.6 min respectively. For linearity seven-point calibration curves were obtained in a concentration range from 1-7 µg/ml for aspirin, rosuvastatin and clopidogrel with correlation coefficient 0.999, 0.9989, 0.9988 respectively. The high recovery values (99%-101%) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveal that the method is precise. In the present study stability indicating an RP-HPLC method for the combination was tested by degrading the drugs together under various stress condition like acid, base and neutral hydrolysis, oxidation, thermal and photolytic stress which is recommended by ICH.Conclusion: The developed RP-HPLC method is simple, economic, specific, accurate and precise for the simultaneous estimation of aspirin, rosuvastatin, and clopidogrel in the combined capsule dosage form. The developed stability indicating analytical method can be used to check the stability of the compounds and was found suitable to determine % degradation of drugs in pharmaceutical dosage form.

Analytical method development of methylisothiazolinone, a preservative, in rat plasma using LC-MS/MS

Methylisothiazolinone (MI) is a preservative used in consumer products to control bacterial and fungal growth. MI can be toxic, act as a skin sensitizer and irritant, and initiate lung diseases; therefore, it is important to understand the mechanisms underlying the toxicity of MI in the body. In this study, we developed a method to analyze plasma MI by using an LC-MS/MS-coupled multiple reaction monitoring (MRM) technique, which follows the fragments of a target metabolite in rat plasma. The MRM transition of MI was m/z 116 ➔ 101, and the lower limit of quantification (LLOQ) was set at 10 ng/mL. Including the concentration of LLOQ, a seven-point calibration curve explained much of the variation in the response, and it was strongly linear (R2 = 0.9998); its intra- and inter-day accuracy and precision values were within 15% of the standard deviation (SD%) and along with the FDA and Korea Ministry of Food and Drug Safety guidelines. For intravenous (iv) pharmacokinetic studies of MI using rats, we developed an analytical method that was useful in detecting the profile of MI in the plasma. We also determined half-life, and area under the curve (AUC) of MI using a non-compartment model, and these might be useful for the study of toxic mechanisms of MI in the body.

Determination of urinary trans,trans-muconic acid using molecularly imprinted polymer in microextraction by packed sorbent followed by liquid chromatography with ultraviolet detection.

trans,trans-Muconic acid (tt-MA) has been introduced as biological exposure index for workers exposed to benzene. In the present study, for the first time, a novel and easy-to-use analytical method based on molecularly imprinted polymer (MIP) in microextraction by packed sorbent (MEPS), followed by high-performance liquid chromatography with ultra-violet detection (MIMEPS-HPLC-UV) was developed to determine tt-MA in urine samples. Fourier transform infrared spectroscopy was used for MIP characterization. The imprinting effect on the MIP was also evaluated. Furthermore, various parameters that affect the MEPS performance were investigated, including extraction cycles, sample volume, as well as the type and volume of washing and elution solvents. The optimised procedure was selective, sensitive, rapid and both user- and environmentally-friendly. A seven-point calibration curve was obtained in the range of 0.015-2μg/mL (R2=0.9986). The extraction recovery was >89.8%. The limit of detection and limit of quantitation for tt-MA were 0.015 and 0.05μg/mL, respectively. The within- and between-day precisions, given as percent relative standard deviation, were in the range of 3.4-4.7% and 3.5-6.6%, respectively. Accuracy was -8.4 to -10.2% in urine samples. The developed method is suggested as an alternative to existing conventional SPE methods for biomonitoring of benzene-exposed subjects.

Simultaneous quantification of methiocarb and its metabolites, methiocarb sulfoxide and methiocarb sulfone, in five food products of animal origin using tandem mass spectrometry.

A simultaneous analytical method was developed for the determination of methiocarb and its metabolites, methiocarb sulfoxide and methiocarb sulfone, in five livestock products (chicken, pork, beef, table egg, and milk) using liquid chromatography-tandem mass spectrometry. Due to the rapid degradation of methiocarb and its metabolites, a quick sample preparation method was developed using acetonitrile and salts followed by purification via dispersive- solid phase extraction (d-SPE). Seven-point calibration curves were constructed separately in each matrix, and good linearity was observed in each matrix-matched calibration curve with a coefficient of determination (R2) ≥ 0.991. The limits of detection and quantification were 0.0016 and 0.005mg/kg, respectively, for all tested analytes in various matrices. The method was validated in triplicate at three fortification levels (equivalent to 1, 2, and 10 times the limit of quantification) with a recovery rate ranging between 76.4-118.0% and a relative standard deviation≤10.0%. The developed method was successfully applied to market samples, and no residues of methiocarb and/or its metabolites were observed in the tested samples. In sum, this method can be applied for the routine analysis of methiocarb and its metabolites in foods of animal origins.

A Rapid and Sensitive UPLC-MS/MS-Method for the Separation and Quantification of Branched-Chain Amino Acids from Dried Blood Samples of Patients with Maple Syrup Urine Disease (MSUD)

Newborn screening for MSUD is a special challenge since patients with MSUD can metabolically decompensate rapidly without adequate treatment within the first two weeks of life. However, the screening method does not detect the actual marker metabolite (alloisoleucine) specifically, but only as part of the group of the other isobaric amino acids leucine, isoleucine and hydroxyproline. We describe a sensitive and rapid second-tier UPLC-MS/MS method to determine branched-chain amino acids from the initial extraction of the screening sample. Quantification is based on a seven-point calibration curve. Reference ranges (mean ± SD in µmol/L) were determined from 179 normal, not pre-selected samples from the newborn screening: leucine: 72 ± 27; isoleucine: 37 ± 19; valine: 98 ± 46; hydroxyproline: 23 ± 13. The concentration of alloisoleucine was below the detection limit in about 55% of the cases, and the highest concentration was 1.9 µmol/L. In all 30 retrospectively studied screening samples from patients with confirmed MSUD the concentration of alloisoleucine was significantly increased. In 238 samples with false-positive newborn screening due to a significant increase in the combined concentration of leucine + isoleucine + alloisoleucine + hydroxyproline (400 to &gt;4000 µmol/L), alloisoleucine was below 6.5 µmol/L (n = 57) or not detectable (n = 181). The application of this assay markedly reduces the false-positive rate and the associated anxiety and costs. It is also suitable for routinely monitoring blood spots of patients with MSUD.

Quantitative, Multidrug Pain Medication Testing by Liquid Chromatography: Tandem Mass Spectrometry (LC-MS/MS).

Chronic pain is often treated with narcotic analgesics. The most commonly used narcotic analgesics are the opiates (natural or modified compounds of the poppy plant) or opioids (synthetic chemicals that act on opiate receptors). While opiates and opioids are excellent analgesics, they can also have significant side effects that include respiratory depression, coma, or death. Tolerance, physical dependence, and addiction (psychological dependence) are other severe side effects of opioid use. Patients who develop dependence or addiction often times abuse other, non-opioid narcotics and may trade their prescription medication for illegal street drugs (called "diversion"). In order to minimize side effects, detect possible multidrug abuse and prove diversion, simultaneous monitoring of numerous prescription and illicit drugs is required. The method described in this chapter is for the quantitative measurement of 43 different drugs in urine. The panel includes narcotic pain medications, benzodiazepines, NIDA drugs, and other, commonly abused medications. The analytes of interests are injected in the presence of deuterated internal standards to correct for possible extraction inefficiencies, ion suppression, or other interferences. The sample is prepared by adding dilution buffer with the deuterated internal standards to the sample, followed by reversed-phase, gradient HPLC separation on a Phenyl-Hexyl column using water and methanol as mobile phases. Detection of the analytes of interest is done by isotope-dilution mass spectrometry on a triple-quadrupole tandem mass spectrometer following electrospray ionization in the positive mode. Mass spectrometric (MS) data are collected in the scheduled MRM (sMRM) mode. Two MRM transitions are monitored for each analyte and one MRM transition is monitored for each IS. Quantitation of the unknown analytes is achieved by comparing the peak area ratios of the analytes to that of the internal standards and reading the unknown concentration from a seven-point calibration curve.

Dried blood spot analysis of creatinine with LC-MS/MS in addition to immunosuppressants analysis

In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was used for the analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in transplant patients with the use of Whatman FTA DMPK-C cards. The method was validated using three different strategies: a seven-point calibration curve using the intercept of the calibration to correct for the natural presence of creatinine in reference samples, a one-point calibration curve at an extremely high concentration in order to diminish the contribution of the natural presence of creatinine, and the use of creatinine-[(2)H3] with an eight-point calibration curve. The validated range for creatinine was 120 to 480 μmol/L (seven-point calibration curve), 116 to 7000 μmol/L (1-point calibration curve), and 1.00 to 400.0 μmol/L for creatinine-[(2)H3] (eight-point calibration curve). The precision and accuracy results for all three validations showed a maximum CV of 14.0% and a maximum bias of -5.9%. Creatinine in DBS was found stable at ambient temperature and 32 °C for 1 week and at -20 °C for 29 weeks. Good correlations were observed between patient DBS samples and routine enzymatic plasma analysis and showed the capability of the DBS method to be used as an alternative for creatinine plasma measurement.

Performance evaluation of a rapid‐scanning quadrupole mass spectrometer in the comprehensive two‐dimensional gas chromatography analysis of pesticides in water

The performance of a novel rapid-scanning (20,000 amu/s) quadrupole mass spectrometer (qMS) has been evaluated in the comprehensive 2-D gas chromatography (GC×GC) analysis of pesticides contained in water. Analyte extraction was performed by using direct solid-phase microextraction (SPME). The MS system was operated using a rather wide m/z 50-450 mass range and a 33 Hz spectral production rate, a frequency which was found sufficient for reliable quantification. The qMS performance was evaluated considering: (i) number of data points per peak, (ii) mass spectral quality, (iii) extent of peak skewing, and (iv) consistency of retention times. Seven-point calibration curves (external calibration) were constructed for 28 pesticides over the limit of quantification range of 100 μg/L (1, 5, 10, 25, 50, and 100 μg/L). The solid-phase microextraction-GC×GC-qMS method was validated by calculating limits of detection and quantification, intraday peak area precision, accuracy, and intraday retention-time precision. A series of tap water samples were subjected to analysis, fortunately giving negative results.

Estimation of blood alcohol concentration by horizontal attenuated total reflectance–Fourier transform infrared spectroscopy

Numerous methods like distillation followed by iodometric titrations, gas chromatograph (GC)–flame ionization detector, gas chromatograph–mass spectrophotometer, GC-Headspace, Breath analyzer, and biosensors including alcohol dehydrogenase (enzymatic) have been used to determine blood alcohol concentration (BAC). In the present study, horizontal attenuated total reflectance–Fourier transform infrared spectroscopy had been used to determine BAC in whole blood. The asymmetric stretching frequency of C–C–O group of ethanol in water (1,045 cm −1) had been used to calculate BAC using Beer's Law. A seven-point calibration curve of ethanol was drawn in the concentration range 24–790 mg dL −1. The curve showed good linearity over the concentration range used ( r 2 = 0.999, standard deviation = 0.0023). The method is accurate, reproducible, rapid, simple, and nondestructive in nature.

Quantitative chiral analysis of phthaloylglutamic acid and related analogs by a single ratio kinetic method using electrospray ionization and matrix‐assisted laser desorption techniques

A rapid method for quantitative chiral analysis of phthaloylglutamic acid and its dimethyl ester by Cook's kinetic method is demonstrated using electrospray ionization (ESI) and matrix-assisted laser desorption techniques. Transition-metal-bound complex ions containing the chiral phthaloylglutamic acid and its dimethyl ester are generated by ESI mass spectrometry and subjected to collision-induced dissociation. The ratio of the two competitive dissociation rates is related to the enantiomeric composition of the drug mixture. A seven-point calibration curve, derived from the kinetic method, allowed rapid quantitation of the enantiomeric excess of drug mixtures. In this paper, matrix-assisted laser desorption/ionization (MALDI) coupled with the linear ion trap (LIT) technique is evaluated for its applicability as a complementary technique to ESI for chiral discrimination and quantitation.

Separation and quantitation of phycobiliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection

The similar electrophoretic mobilities and sizes of several of the phycobiliproteins, which are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae, render their separation and quantitation a challenging problem. However, we have developed a suitable capillary electrophoresis (CE) method that employs a phytic acid–boric acid buffer and laser-induced fluorescence (LIF) detection with a single 594 nm He–Ne laser. This method takes advantage of the remarkably high quantum yields of these naturally fluorescent proteins, which can be attributed to their linear tetrapyrrole chromophores covalently bound to cysteinyl residues. As such, limits of detection of 1.18·10 −14, 5.26·10 −15, and 2.38·10 −15 mol/l were obtained for R-phycoerythrin, C-phycocyanin, and allophycocyanin proteins, respectively, with a linear dynamic range of eight orders of magnitude in each case. Unlike previously published CE–LIF methods, this work describes the separation of all three major classes of phycobiliproteins in under 5 min. Very good recoveries, ranging from 93.2 to 105.5%, were obtained for a standard mixture of the phycobiliproteins, based on seven-point calibration curves for both peak height and peak area. It is believed that this development will prove useful for the determination of phycobiliprotein content in naturally occurring cyanobacteria populations, thus providing a useful tool for understanding biological and chemical oceanographic processes.