Preparation Of Calibration Standards Research Articles

Liquid Chromatography/Tandem Mass Spectrometry Method for Estimation of Cholic Acid in Rat Plasma, Urine and its Application

Cholic acid is a primary bile acid synthesised from cholesterol in liver. Apart from being major catabolic products of cholesterol bile acids facilitates absorption of fats in intestine. A selective, sensitive MRM based liquid chromatography/ tandem mass spectrometry method has been developed and validated for the estimation of cholic acid in rat plasma and urine. Telmisartan was used as internal standard (IS). Electrospray ionization (ESI) probe with negative mode of operation was used for the ionisation of cholic acid and IS. Chromatographic separation was performed through X-bridge C18 column with the mobile phase of 0.1% formic acid in water (aqueous reservoir), 100% methanol (organic modifier). A short LC gradient of 3 minutes run time was used with flow rate of 0.7 mL/min. Charcoal stripped plasma and urine were used for the preparation of calibration standards and quality control samples. The analyte and IS were isolated from plasma and urine by a simple organic solvent based protein precipitation. The assay was linear in the concentration range of 31.26-10000 ng/mL. The method has been validated according to published FDA guidelines and showed excellent performance. The developed method was applied for the estimation of cholic acid in plasma and urine samples from liver toxicity experiment in rats. Liver toxicity was induced by intraperitoneal injection of carbon tetrachloride (CCl4).

A novel strategy for preparing calibration standards for the analysis of plant materials by laser-induced breakdown spectroscopy: A case study with pellets of sugar cane leaves

Calibration is still a challenging task when dealing with the direct analysis of solids. This is particularly true for laser-induced breakdown spectroscopy (LIBS), and laser ablation inductively coupled plasma optical emission spectrometry/mass spectrometry, when the calibrations are matrix-dependent and/or appropriate certified reference materials are generally not available. Looking at the analysis of plant materials in the form of pressed pellets by LIBS, a new method to overcome and/or minimize this difficulty is proposed by keeping the matrix constant in order to produce matrix-matched calibration pellets. To achieve this goal and to test this novel approach, ground sugar cane leaves were chosen and submitted to acid extractions for obtaining the corresponding blank or a material containing very low concentrations of the analytes. The resulting dried solid material was used either as a blank or a low concentration standard, and also homogeneously mixed with the original plant material at appropriate ratios as well. The corresponding pellets were used as calibration standards and ablated at 30 different sites by applying 25 laser pulses per site with a Q-switched Nd:YAG at 1064nm. The plasma emission collected by lenses was directed through an optical fiber towards a spectrometer equipped with Echelle optics and intensified charge-coupled device. Delay time and integration time gate were fixed at 2.0 and 5.0μs, respectively. This calibration strategy was tested for the determination of Ca, Mg, K, P, Cu, Mn, and Zn by LIBS in pellets of leaves from 17 varieties of sugar cane and good correlations were obtained with inductively coupled plasma optical emission spectrometry results in the corresponding acid digests. The proposed approach was also useful to estimate the limits of detection based on measurements of blanks, as recommended by IUPAC, or with the aid of a low concentration standard.

Quantitation by GC–MS of Methylglyoxal as a Marker in Anxiety-Related Studies

Methylglyoxal (MG) is present in the human system. In the cell, MG is degraded by glyoxalase 1 (Glo 1), an enzyme differentially expressed in several mouse models of anxiety-related behavior. It has also been associated with the formation of advanced glycation and lipoxidation end products, contributing to diabetes-related complications. Approaches developed for accurate quantitation of MG in this study include the synthesis of a deuterated MG analog to serve as an internal standard; skillful application of the standard addition protocol (with “reiteration”) to quantitate MG in “blank” samples, allowing the preparation of calibration standards using the same test specimen matrix system; and various pretreatment methods, including extraction and derivatization, to prepare complex brain tissue specimens for gas chromatography–mass spectrometry analysis. Using our method, brain MG levels in high- and low-anxiety-related behavior (HAB and LAB) mice were determined as 5.11 ± 0.89 and 13.05 ± 0.89 μg g−1, respectively. These data supported the conclusion that MG level in brain is negatively correlated with anxiety in mouse models of anxiety-related studies, contributing to better understanding of the molecular mechanisms underlying reported observations.

Matrix effects in analysis of dialkyl phosphate metabolites of organophosphate pesticides in urine by gas chromatography/tandem mass spectrometer

Urinary dialkyl phosphate metabolites (DAPs) are used as biomarkers to evaluate human exposure to organophosphate pesticides. The objective was to evaluate potential artifacts in urinary DAPs analysis during sample preparation and method calibration. Diluted urine pools were commonly used to prepare calibration standards to minimize the effects due to the complexity of urine matrix. Matrix effects on measurements of DAPs were evaluated by spiking known amount of standards into distilled water, synthetic urine and diluted urine pool. Different matrices resulted in similar concentrations detected for all target compounds, except dimethylphosphate (DMP) with the deviation of measurement as large as eight times the spiked amount. In this study, we also found that urinary particles, which usually appeared after thawing frozen human urine samples, could affect the measurements of DAPs, especially DMP and diethylthiophosphate (DETP). Results of DAPs measurements in three types of sample matrices, i.e. urine without particles, urine with particles and particles only were compared. DETP could be subject to large error during this preparation step. The use of deuterated and 13C12-labeled DAPs as internal standards is also evaluated. Overall, these issues can cause misidentification and inaccuracies, which may significantly affect the data quality.

Determination of Platinum Originating from Carboplatin in Human Urine and Canine Excretion Products by Inductively Coupled Plasma Mass Spectrometry

We present highly sensitive, rapid methods for the determination of Pt originating from carboplatin in human urine and canine urine, feces, and oral fluid. The methods are based on the quantification of Pt by inductively coupled plasma mass spectrometry, and allow quantification of 7.50 ng/L Pt in human and canine urine (in 15 μL of matrix), 15.0 ng/L Pt in canine oral fluid (in 15 μL of matrix), and 0.105 ng/g Pt in canine feces (in 5 μg of matrix). Sample pretreatment mainly involved dilution with appropriate diluents. The performance of the methods fulfilled the most recent FDA guidelines for bioanalytical method validation. Validated ranges of quantification were 7.50 to 1.00 × 10(4) ng/L Pt in human and canine urine, 0.105-30.0 ng/g Pt in canine feces, and 15.0 to 1.00 × 10(4) ng/L Pt in canine oral fluid. Canine urine and oral fluid cannot be easily obtained. Therefore, we also investigated the validity of the usage of human matrix samples for the preparation of calibration standards and quality control samples as alternatives, to be used in future clinical studies. The assays are used to support biomonitoring studies and pharmacokinetic studies in pet dogs treated with carboplatin.

Coupling Desorption Electrospray Ionization with Solid-Phase Microextraction for Screening and Quantitative Analysis of Drugs in Urine

Direct analysis of silica C(18)-coated solid-phase microextraction (SPME) fibers using desorption electrospray ionization mass spectrometry (DESI-MS) for the purpose of analyzing drugs from raw urine is presented. The method combines a simple, inexpensive, and solvent-less sample preparation technique with the specificity and speed of DESI-MS and MS/MS. Extraction of seven drugs from raw urine is performed using specially designed SPME fibers coated uniformly with silica-C(18) stationary phase. Each SPME device is inserted into unprocessed urine under gentle agitation and, then, removed, rinsed, and analyzed directly by DESI-MS (MS/MS). Rapid screening over a wide mass range is afforded by coupling the method with a time of flight (TOF) mass spectrometer while quantitative analysis is performed using selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer. The performance of the SPME DESI-MS/MS method was evaluated by preparing calibration standards and quality control (QC) samples of the seven drug compounds from urine over a range from 20 to 1000 ng/mL, with the exception of meprobamate which was prepared from 200 to 10000 ng/mL. The calibration curves constructed for each analyte had an R(2) > 0.99. The range of precision (%CV) and accuracy values (% bias) for low QC samples was 1-11% and 3-38%, respectively. Precision and accuracy values for high QC samples range from 0.9 to 8% and -31 to -8%. Results from urine specimens of actual exposure to drugs screened using the SPME DESI-MS/MS method showed good agreement with the conventional immunoassays and GC/MS analysis. Liquid desorption of the SPME fiber followed by LC/MS/MS also showed good agreement with the SPME DESI-MS/MS method.

Development and Validation of a Selective and Robust LC-MS/MS Method, Employing Liquid–Liquid Extraction Based on 96-Well Format Plates, for High-Throughput Quantification of Docetaxel in Small Plasma Samples

To investigate the pharmacokinetic characteristics of a novel nanoliposome-entrapped docetaxel injection, an anti-tumor agent, a high-throughput analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of docetaxel in beagle dog plasma using paclitaxel as the internal standard (I.S.). Most liquid transfer steps, including preparation of calibration standards and quality control samples, as well as the addition of the I.S., were performed by using eight-channel pipetting tools. The analyte and internal standard were isolated from 50 µL plasma samples and chromatographed on a Ultimate XB C-8 column (50 mm × 4.6 mm, 5 µm) with a mobile phase consisting of methanol–water–formic acid (90:10:0.01, v/v/v)pumped at 0.3 mL/min. The method had a chromatographic total run time of 3 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 830.0 → 549.0 (docetaxel) and 876.0 → 591.0 (paclitaxel) used for quantitation. The method was sensitive, with a lower limit of quantitation (LLOQ) of 1 ng/mL and good linearity in the range 1–500 ng/mL for docetaxel. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to initial pharmacokinetic study of nanoliposome-entrapped docetaxel injection in beagle dogs, and the statistical results of the pharmacokinetic data clearly suggest the novel formulation vehicle does control docetaxel's acute side effects to some extent.

Sensing bacteria but treating them well: Determination of optimal incubation and storage conditions

Bacteria detection in real samples often involves long and tedious methodologies such as culture enrichment, biochemical screening, and serological confirmation. In this context, the development of biosensors and quick assays for bacteria detection appears as fast growing fields. However, a detailed study of reports in these areas reveals the existence of important differences in bacteria storage, handling, and detection conditions, indicating that authors do not take advantage of the well-established procedures existing for classical techniques such as enzyme-linked immunosorbent assay (ELISA). In the current work, we exploit standard ELISA methodology to identify and study diverse parameters that can be critical along the different steps of bacteria detection and sensing. Among others, we studied in detail the effect of the bacterial strain used and the presence of detergent and glycerol in assay performance, as well as the effects of heat inactivation or storing conditions, on bacteria integrity and thus detectability. Finally, we describe the use of “ready-to-use” frozen bacterial pellets as an excellent alternative to the use of daily prepared fresh cultures during assay optimization and preparation of calibration standards. The results presented are also supported by an extensive bibliography search, giving shape to an important compilation of information that will be useful to authors working in a variety of methodologies and sensing formats.

A novel derivatization approach for simultaneous determination of glyoxal, methylglyoxal, and 3-deoxyglucosone in plasma by gas chromatography–mass spectrometry

A two-step derivatization approach has been developed to enable the simultaneous analysis of glyoxal, methylglyoxal, and 3-deoxyglucosone by the most efficient and widely applied GC–MS methodology. These three analytes are reactive carbonyl compounds associated with the formation of advanced glycation and lipoxidation end products, a process thought to contribute to uremic toxicity and referred to as “carbonyl stress”. Effective analysis of these compounds would facilitate understanding these compounds’ role in diabetes-related complications. Plasma samples were deproteinized by acetonitrile, followed by a two-step derivatization approach. Pooled plasma samples from healthy individuals were used as the “blank” for preparing calibration standards. The concentrations of the analytes in the “blank” were first determined by standard addition method. Calibration parameters were accordingly established and used to analyze these compounds in plasma samples collected from healthy individuals and diabetic patients. Analytical findings are comparable with those reported in the literature. Quantitation data can be further improved by making available and using isotopically labeled analogs of these analytes as the internal standards.

A Study of the Critical Uncertainty Contributions in the Analysis of PCBs in Ambient Air

The measurement of polychlorinated biphenyls (PCBs) in ambient air requires a complex, multistep sample preparation procedure prior to analysis by gas chromatography—mass spectrometry (GC-MS). Although routine analytical laboratories regularly carry out these measurements, they are often undertaken with little regard to the accurate calculation of measurement uncertainty, or appreciation of the sensitivity of the accuracy of the measurement to each step of the analysis. A measurement equation is developed for this analysis, and the contributory sources to the overall uncertainty when preparing calibration standards and other solutions by gravimetric and volumetric approaches are discussed and compared. For the example analysis presented, it is found that the uncertainty of the measurement is dominated by the repeatability of the GC-MS analysis and suggested that volumetric (as opposed to gravimetric) preparation of solutions does not adversely affect the overall uncertainty. The methodology presented in this work can also be applied to analogous methods for similar analytes, for example, those used to measure polycyclic aromatic hydrocarbons (PAHs), pesticides, dioxins, or furans in ambient air.

Estimation of uncertainty in electron probe microanalysis: iron determination in manuscripts, a case study

A case study of ISO GUM-based estimation of measurement uncertainty of quantitative surface elemental analysis is presented. The analytical task was the measurement of iron content in the ink writing on the surface of an 18th century manuscript by electron probe microanalysis using a scanning electron microscope equipped with an energy-dispersive X-ray spectrometer (SEM/EDS). The problems that arise in uncertainty estimation of quantitative surface analysis are outlined (defining the measurand, preparing calibration standards, non-uniformity of the surface, etc) and ways of overcoming them are suggested. The average iron content on the ink-covered surface of the sample manuscript was found to be 0.12 ± 0.04 µg mm−2 (at confidence level 95.5%). Paper sheets with ink lines of known iron contents were used as calibration standards. The main source of uncertainty was the variability of the parallel measurement results from different locations of the sample surface (mostly due to the variations in the surface structure of paper), which contributed 78% of the total uncertainty. It is concluded that EPMA using a SEM/EDS is suitable for at least semi-quantitative determination of iron in the writing of ink-written manuscripts. Several specific issues in uncertainty analysis are pointed out that need further investigation.

Calorimetric determination of amorphous content in lactose: A note on the preparation of calibration curves

Calorimetric methods (isothermal or solution calorimetry) offer the ability to detect amorphous contents to 0.5% (w/w) or better in processed pharmaceuticals and calorimetric data are becoming more widely accepted in regulatory submissions. However, both methods require the construction of calibration curves, prepared using quantitative physical mixtures of entirely amorphous and entirely crystalline material. If the sample under investigation exists in two or more isomers or polymorphs, and the enthalpy of solution (solution calorimetry) or the enthalpy of crystallisation (isothermal calorimetry) are different for the isomers or polymorphs, then it must be ensured that the batch of material used to prepare the calibration samples has the same isomeric or polymorphic composition as the (processed) material to be tested. Here, we demonstrate the problems that may arise using lactose as a pharmaceutically important model substance. Calibration curves were prepared from solution calorimetry and isothermal gas perfusion calorimetry data using two batches of lactose (one predominantly anhydrous α-lactose and one predominantly β-lactose). The calibration curves are shown to be significantly different for the two batches, and it is shown that quantification of the amorphous content of a processed sample of unknown isomeric composition is impossible, unless the calibration curve is prepared from the same batch of material as the processed sample. In addition, some of the other problems inherent in using isothermal gas perfusion calorimetry for amorphous content determination, such as wetting issues and the preparation of calibration standards that mimic processes samples, are discussed.

Determination of VOC and TVOC in Air Using Thermal Desorption GC-MS – Practical Implications for Test Chamber Experiments

The determination of volatile organic compounds (VOC) species and concentrations are important for the evaluation of indoor air quality. Numerous methods exist for the determination of levels of both VOC and TVOC (total volatile organic compounds). These include the use of direct-reading instruments as well as gas chromatographic techniques. The benefits and drawbacks of the various methods are well known, and none provide precise measurement of the complex VOC mixtures that constitute TVOC in indoor air. A specific approach for TVOC measurement has been proposed in Europe in ECA-report no.19 to overcome these shortcomings. On this basis a practical analytical method was developed and applied to emission test chamber studies. Special focus was required on compounds of high volatility and the preparation of calibration standards.

Laboratory and Field Evaluation of a SAW Microsensor Array for Measuring Perchloroethylene in Breath

This article describes the laboratory and field performance evaluation of a small prototype instrument employing an array of six polymer-coated surface acoustic wave (SAW) sensors and a thermal desorption preconcentration unit for rapid analysis of perchloroethylene in breath. Laboratory calibrations were performed using breath samples spiked with perchloroethylene to prepare calibration standards spanning a concentration range of 0.1–10 ppm. A sample volume of 250 mL was preconcentrated on 40 mg of Tenax® GR at a flow rate of 100 mL/min, followed by a dry air purge and thermal desorption at a temperature of 200°C. The resulting pulse of vapor was passed over the sensor array at a flow rate of 20 mL/min and sensor responses were recorded and displayed using a laptop computer. The total time per analysis was 4.5 min. SAW sensor responses were linear, and the instrument's limit of detection was estimated to be 50 ppb based on the criterion that four of the six sensors show a detectable response. Field performance was evaluated at a commercial dry-cleaning operation by comparing prototype instrument results for breath samples with those of a portable gas chromatograph (NIOSH 3704). Four breath samples were collected from a single subject over the course of the workday and analyzed using the portable gas chromatograph (GC) and SAW instruments. An additional seven spiked breath samples were prepared and analyzed so that a broader range of perchloroethylene concentrations could be examined. Linear regression analysis showed excellent agreement between prototype instrument and portable GC breath sample results with a correlation coefficient of 0.99 and a slope of 1.04. The average error for the prototype instrument over a perchloroethylene breath concentration range of 0.9–7.2 ppm was 2.6% relative to the portable GC. These results demonstrate the field capabilities of SAW microsensor arrays for rapid analysis of organic vapors in breath.

High-throughput sample preparation procedures for the quantitation of a new bone integrin α νβ 3 antagonist in human plasma and urine using liquid chromatography–tandem mass spectrometry

High throughput LC–MS/MS assays to quantitate a new α νβ 3 bone integrin antagonist ( I) in human plasma and urine have been developed using instruments programmed to automate sample preparation procedures. Packard liquid handling system-MultiPROBE ® II EX was programmed for preparing calibration standards in control plasma and urine, acidifying all standards, quality control (QC), and clinical samples with necessary dilutions, and adding the internal standard to the acidified samples. TOMTEC Quadra 96™ was programmed to perform the solid phase extraction (SPE) process on a 3 M 96-well mixed phase cation standard density (MPC-SD) plate to isolate the analytes from the sample matrix. The extract collected from both types of matrices was directly injected into reversed-phase LC–MS/MS system with a Turbo Ion Spray (TIS) interface in the positive ionization mode. The plasma and urine assays have the calibration range of 0.5–1500 and 2–6000 ng/mL, respectively. Validation of the automated and the manual plasma assays showed that application of MultiPROBE II to sample preparation gave comparable accuracy and precision. Overall, the automated approaches with minimum manual intervention enhanced the throughput of sample preparation.

Matrix effects in the gas chromatographic–mass spectrometric determination of brominated analogues of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone

Brominated analogues (BMXs) of the strong drinking water mutagen MX (3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone) were found to be subject to strong matrix induced chromatographic response enhancement effects. We evaluated different ways to reduce errors in quantification including comparison of gas chromatographic inlet systems, improved clean up of sample extracts, and preparation of calibration standards in the sample matrix. The best quantitative accuracy and long term performance were achieved when the calibration standards were prepared in sample matrix, samples were cleaned up with C 18-resin in conjunction with solid phase extraction (SPE) with Oasis HLB cartridges, and gas chromatography with PTV splitless injection was used. This method enables the determination of MX and BMXs from 500 ml water sample with quantitation limits of 1 ng/l or less.

Quantitative analysis of simvastatin and its β-hydroxy acid in human plasma using automated liquid–liquid extraction based on 96-well plate format and liquid chromatography-tandem mass spectrometry

An assay based on automated liquid–liquid extraction (LLE) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed and validated for the quantitative analysis of simvastatin (SV) and its β-hydroxy acid (SVA) in human plasma. A Packard MultiProbe II workstation was used to convert human plasma samples collected following administration of simvastatin and quality control (QC) samples from individual tubes into 96-well plate format. The workstation was also used to prepare calibration standards and spike internal standards. A Tomtec Quadra 96-channel liquid handling workstation was used to perform LLE based on 96-well plates including adding solvents, separating organic from aqueous layer and reconstitution. SV and SVA were separated through a Kromasil C18 column ( 50 mm×2 mm i.d., 5 μm) and detected by tandem mass spectrometry with a TurboIonspray interface. Stable isotope-labeled SV and SVA, 13 CD 3 -SV and 13 CD 3 -SVA, were used as the internal standards for SV and SVA, respectively. The automated procedures reduced the overall analytical time (96 samples) to 1/3 of that of manual LLE. Most importantly, an analyst spent only a fraction of time on the 96-well LLE. A limit of quantitation of 50 pg/ml was achieved for both SV and SVA. The interconversion between SV and SVA during the 96-well LLE was found to be negligible. The assay showed very good reproducibility, with intra- and inter-assay precision (%R.S.D.) of less than 7.5%, and accuracy of 98.7–102.3% of nominal values for both analytes. By using this method, sample throughput should be enhanced at least three-fold compared to that of the manual procedure.

Measurement of [formula omitted] in dust from steel scrap smelting

This paper deals with the measurement of 137Cs content in dust produced during steel scrap smelting and in the material produced from recycling the dust to recover the zinc oxide component. The measured samples have different densities and chemical compositions. The addition of radioactive standards to this kind of material to prepare a calibration standard is rather difficult. A theoretical analysis of the attenuation coefficient for this material together with its experimental measurement, show a linear relationship between the coefficient and density independent of chemical composition that allows the use of a soil sample to prepare calibration standards. An analysis of the validity of the method, as well as the development of a density correction factor is detailed. This factor is calculated incorporating the linear relationship between attenuation coefficient and density to the integral representing detection efficiency, and evaluating the integral by means of a Monte-Carlo program.

Calibration of laser-ablation ICP-MS. Can we use synthetic standards with pneumatic nebulization?

An approach for the determination of trace element concentrations in high purity metals, using an inductively coupled plasma mass spectrometer (ICP-MS) with a laser-ablation system for direct solid sample introduction after calibration with nebulized liquid standards was made. Due to the inherent differences in the rate of sample introduction with laser-ablation and pneumatic nebulization, a matrix element must be used as an internal standard. This is problematical for elements that have no isotope with a relative abundance of less than 0.1 %, since the ion signals would be too high for direct measurement, and reduction of the ablation rate would compromise the sensitivity for trace elements. Due to the high stability of ICP-unit and mass filter of the instrument used, it was found that the tail of a mass-peak of the matrix element could be used as an internal standard. Therefore, a position at –0.5 amu from the matrix-isotope (e.g. 62.5Cu in copper samples) was used for internal standardization. The standard deviation of this signal in a period of 2.5 h was 3.6% RSD with no notable drift when the laser ablation was used for sample introduction. The calibration of the matrix-element by nebulizing liquid standards showed that the ion signal measured on the peak-tail is directly proportional to the element concentration in the ICP. This indicates that the peak shape is not only stable, but also independent of the peak height. The advantages of this method lie in the easy preparation of calibration standards for quantitative measurements with a laser-ablation system and access to homogeneous standards for materials, that are difficult to homogenize in the solid state. The calibration of the traces is performed relatively to a fixed concentration of the matrix element. Calibrations were carried out for trace concentrations in high purity copper and good recoveries were obtained for high-purity reference standards.

Analysis of individual natural pyrethrins in indoor matrices by HRGC/ECD

AbstractAn HRGC/ECD method is presented which permits the identification and quantitation of individual components of pyrethrins (cinerin I, jasmolin I, pyrethrin I, cinerin II, jasmolin II, pyrethrin II) in indoor matrices (airborne particles, house dust) after application of an agent containing pyrethrum. In order to prepare calibration standards for the individual components of the pyrethrum extract, the pyrethrins were isolated hy semi‐preparative HPLC and their content and purity were determined by 1H‐NMR spectroscopy.