Microderivatization of 4-[O]hydroxyproline and quantitation with a benchtop mass spectrometer

Accurate estimation of in vivo turnover rates of collagen is complicated by amino acid reutilization. It was previously shown that the ideal, non-recycling tracer was [¹⁸O]hydroxyproline synthesized in vivo. The analytical method for measuring turnover rates with [¹⁸O]hydroxyproline must include analyte quantitation for pool size determination and isotope ratio measurement for determining levels of label incorporation. For ease of use and widest availability, a benchtop gas chromatograph—mass spectrometer in the electron-impact ionization mode was chosen. Here we present a versatile procedure for hydroxyproline derivatization that is well suited for routine, large-scale determination of analyte concentrations and relative levels of ¹⁸O incorporation.     

Luminescence catalyst immunoassay of β2-microglobulin with haemin as a chemically amplifiable label

Luminescence catalyst immunoassay, a nonradioactive and nonenzymatic immunoassay, has been applied to the determination of β₂-microglobulin (β₂-MG). The method is characterized by high sensitivity and simple handling, since the analytical method employs a catalytically amplifiable label and solid-phase sandwich technique, respectively. In the first stage, an antibody-immobilized plate is reacted with the analyte (β₂-MG). In the second stage, the bound β₂-MG undergoes successive binding with haemin-labelled antibody. In the last stage, the plate is placed in the luminol-H₂O₂ system to generate luminescence. A calibration curve with a β₂-MG concentration at the midpoint of 100 ng ml^?1 was obtained. The 10 and 90% response levels in the curve are 10 and 1000 ng ml^?1.     

Budesonide quantification by HPLC coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometry. Application to a comparative systemic bioavailability of two budesonide formulations in healthy volunteers

In the present study, a novel, fast, sensitive and robust method to quantify budesonide in human plasma using 3-keto-desogestrel as the internal standard (IS) is described. The analyte and the IS were extracted from human plasma by liquid–liquid extraction (LLE) using ether. Extracted samples were analyzed by high performance liquid chromatography coupled to Atmospheric pressure photoionization tandem mass spectrometry (HPLC–APPI-MS/MS). Chromatography was performed isocratically on a C18, 5 μm analytical column. The temperature of the autosampler was kept at 6 °C and the run time was 4.00 min. A linear calibration curve over the range 7.5–1000 pg ml^?1 was obtained and the lowest concentration quantified was 7.5 pg ml^?1, demonstrating acceptable accuracy and precision. This analytical method was applied in a relative bioavailability study in order to compare a test budesonide 64 μg/dose nasal spray formulation vs. a reference 64 μg/dose nasal spray formulation (Budecort Aqua) in 48 volunteers of both sexes. The study was conducted in an open randomized two-period crossover design and with a one-week washout period. Plasma samples were obtained over a 14 h interval. Since the 90% CI for both C_max, AUC_last and AUC_0-inf were within the 80–125% interval proposed by the Food and Drug Administration and ANVISA, it was concluded that budesonide 64 μg/dose nasal spray was bioequivalent to Budecort Acqua^® 64 μg/dose nasal spray, according to both the rate and extent of absorption.     

Determination of ampicillin sodium using the cupric oxide nanoparticles–luminol–H2O2 chemiluminescence reaction

A simple and sensitive chemiluminescence (CL) method has been developed for the determination of ampicillin sodium at submicromolar levels. The method is based on the inhibitory effect of ampicillin sodium on the cupric oxide nanoparticles (CuO NPs)–luminol–H₂O₂ CL reaction. Experimental parameters affecting CL inhibition including concentrations of CuO NPs, luminol, H₂O₂ and NaOH were optimized. Under optimum conditions, the calibration plot was linear in the analyte concentration range 4.0 × 10^‐7–4.0 × 10^‐6 mol/L. The limit of detection was 2.6 × 10^‐7 mol/L and the relative standard deviation (RSD) for six replicate determinations of 1 × 10^‐6 mol/L ampicillin sodium was 4.71%. Also, X–ray diffraction (XRD) and transmission electron microscopy (TEM) analysis were employed to characterize the CuO NPs. The utility of the proposed method was demonstrated by determining ampicillin sodium in pharmaceutical preparation. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of the vanadium content of protein solutions by electron paramagnetic resonance spectroscopy

A rapid and precise method of determining the vanadium content of protein solutions by electron paramagnetic resonance spectroscopy is reported The method is based on the linear relationship between the signal intensity of the first-derivative electron paramagnetic resonance signal and the concentration of the VO(H₂O)₅²⁺ species formed in acid solution. Six different proteins were investigated in the concentration range 10^?3-10^?5m. A precision of ±1%, an accuracy of ±3%, and a detection limit of 25ppb for vanadium was obtained. This analytical method was developed to aid in investigations of metal binding sites in proteins by vanadyl ion (VO²⁺) electron paramagnetic resonance spectroscopy.     

Ultrafast liquid chromatographic analysis of erdosteine in human plasma based on fluorimetric detection and precolumn derivatization with 4‐bromomethyl‐7‐methoxycoumarin: Application to pharmacokinetic studies

In this study, a new analytical method for erdosteine (ERD) in plasma based on high‐performance liquid chromatography and a fluorimetric detector, is presented. Precolumn derivatization of ERD with 4‐bromomethyl‐7‐methoxy coumarin (BrMmC) and dibenzo‐18‐crown‐6‐ether as a reaction catalyst led to the production of a fluorescent compound. ERD was monitored by fluorescence with an excitation wavelength λ_ext. = 325 nm and emission wavelength λ_em. = 390 nm. Optimum reaction conditions were carefully studied and optimized. A chromatographic procedure was performed using a C18 column of 150 × 4.6 mm and 3 μm particle size and a mobile phase consisting of methanol:acetonitrile:water (30:30:40, v/v/v) under a flow rate of 0.5 ml min^?1. A calibration plot was established covering analyte concentration range 0.2–3.0 μg ml^?1; the detection limit was 0.015 μg ml^?1 and quantification limit was 0.05 μg ml^?1. Mean recovery was 87.33% and relative standard deviation was calculated to be less than 4.4%. The developed method was successfully used to determine pharmacokinetic preparations of ERD subsequent to administration of a 900 mg dose capsule to a healthy 40‐year‐old woman volunteer.     

Acoustic-resonance spectrometry as a process analytical technology for the quantification of active pharmaceutical ingredient in semi-solids

The purpose of this study was to demonstrate acoustic resonance spectrometry (ARS) as an alternative process analytical technology to near infrared (NIR) spectroscopy for the quantification of active pharmaceutical ingradient (API) in semi-solids such as creams, gels, ointments, and lotions. The ARS used for this research was an inexpensive instrument constructed from readily available parts. Acoustic-resonance spectra were collected with a frequency spectrum from 0 to 22.05 KHz. NIR data were collected from 1100 to 2500 nm. Using 1-point net analyte signal (NAS) calibration, NIR for the API (colloidal oatmeal [CO]) gave anr ² prediction accuracy of 0.971, and a standard error of performance (SEP) of 0.517%CO. ARS for the API resulted in anr ² of 0.983 and SEP of 0.317%CO. NAS calibration is compared with principal component regression. This research demonstrates that ARS can sometimes outperform NIR spectrometry and can be an effective analytical method for the quantification of API in semi-solids. ARS requires no sample preparation, provides larger penetration depths into lotions than optical techniques, and measures API concentrations faster and more accurately. These results suggest that ARS is a useful process analytical technology (PAT). Published: July 14, 2006     

Determination of the Biomasses of Small Bacteria at Low Concentrations in a Mixture of Species with Forward Light Scatter Measurements by Flow Cytometry

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and ¹⁴C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4′,6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 μg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10³ cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.     

Simple high-performance liquid chromatographic method to analyse megazol in human and rat plasma

A simple and sensitive high-performance liquid chromatographic method has been developed to measure megazol in human plasma. The method was optimized and validated according to the Washington Concensus Conference on the Validation of Analytical Methods (V.P. Shah et al., Eur. J. Drug Metab. Pharmacokinet., 15 (1991) 249). The criteria of complete validation were specificity, linearity, precision, analytical recovery, dilution and stability. It involved extraction of the plasma with dichloromethane, followed by reversed-phase high-performance liquid chromatography using a Kromasil^R C₈ column and UV detection at 360 nm. The retention times of the internal standard (tinidazol) and megazol were 6.10 and 9.60 min, respevtively. The standard curve was linear from 2 ng ml⁻¹ (limit of quantification) to 2000 ng ml⁻¹. The coefficients of variation for all the criteria of validation were less than 6%; 85 to 92% extraction efficiencies were obtained. Megazol was stable during the storage period (one month at −20°C) in plasma and for two months at 25°C in standard solution. The method was tested by measuring the plasma concentration following oral administration to rat and was shown to be suitable for pharmacokinetic studies.     

Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay

We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu²⁺ in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time–response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample.     

Immunoprecipitation on magnetic beads and liquid chromatography-tandem mass spectrometry for carbonic anhydrase II quantification in human serum

In this study, a magnetic bead-based platform amenable to high-throughput protein carbonic anhydrase II (CA II) capture is presented. The key steps in this approach involved immunoaffinity purification of the target protein from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) operating in multiple reaction monitoring acquisition mode. Using a synthetic peptide standard and a structural analogue free-labeled internal standard, the resulting concentration was stoichiometrically converted to CA II serum concentration. The analytical steps, such as preparation of immunobeads, protein capture, proteolysis, and calibration, were optimized. The method was validated in terms of recovery (77%), reproducibility (relative standard deviation [RSD] < 12%), and method detection limit (0.5 pmol ml⁻¹). The developed method was applied to determining the CA II in eight healthy subjects, and the concentration measured was 27.3 pmol ml⁻¹ (RSD = 65%).     

Radiometric determination of cytidine 5′-triphosphate in biological materials

A simple and sensitive radioenzymic method is described for the direct determination of cytidine 5′-triphosphate (CTP) in the range of 1 to 9 nmol in perchloric acid extracts of tissue. The method involves a specific conversion of CTP and [1,2-¹⁴C]phosphorylethanolamine to labeled CDP-ethanolamine using purified CTP:phosphorylethanolamine cytidylyltransferase (EC 2.7.7.14). The [¹⁴C]CDP-ethanolamine formed is isolated on a Dowex 1-formate column taking advantage of the selectivity of borate complexing. A spectrophotometric method for determining CTP in standard solutions is also described.     

Determination of 22-oxacalcitriol, a new analog of 1α,25-dihydroxyvitamin D3, in human serum by liquid chromatography–mass spectrometry

A sensitive and specific liquid chromatographic–mass spectrometric assay has been developed for the determination of 22-oxacalcitriol (OCT), which is a new analog of 1α,25-dihydroxyvitamin D₃. The analyte was isolated from serum by two solid-phase extraction steps on a C₁₈ cartridge and NH₂ cartridge. The recovery of OCT through two extraction steps was more than 90%. A related substance (ED-94), i.e. OCT with the side-chain shortened by one carbon, was used as an internal standard. Extracts were chromatographed on a C₁₈ reversed-phase column interfaced to the electrospray ionization source. The mass spectrometer was operated in the positive-ion mode of selected reaction monitoring. The chromatographic run-time for one injection was less than 6 min. The intra- and inter-assay coefficients of variation for the lowest concentration examined (30 pg ml⁻¹) were 9.83 and 10.67, respectively. And the analytical recovery of OCT added to serum was quantitative. Assay linearity was obtained in the range of 20–640 pg ml⁻¹.     

Pharmacokinetic applicability of a validated liquid chromatography tandem mass spectroscopy method for orally administered curcumin loaded solid lipid nanoparticles to rats

A simple and sensitive validated LC–MS/MS analytical method was used for determination of curcumin in rat plasma, using nimesulide as internal standard. Analyses were performed on an Agilent LC–MS/MS system using a Chromolith rod? and isocratic elution with acetonitrile:10 mM ammonium acetate buffer (pH 3.5) (80:20, v/v) at a flow rate of 0.8 ml/min with a total run time of 3 min and an overall recovery of 77.15%. A triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the negative mode was used. Calibration curve in plasma spiked with varying concentration of curcumin were linear over the concentration range of 10–2000 ng/ml with determination coefficient >0.99. The lower limit of quantification was 10 ng/ml. Intra and inter-day variability's (RSD) for extraction of curcumin from plasma were less than 10% and 15% respectively and accuracy was 102.43–108.5%. Multiple reaction monitoring was used to monitor the transition for curcumin (m/z; 367/217 [M?H]^?) and IS (m/z; 307/229). The method was applied for determining curcumin concentration in plasma after peroral administration of 50 mg/kg of free curcumin (C-S) or curcumin loaded solid lipid nanoparticles (C-SLNs) to rats. Results established selectivity and suitability of the method for pharmacokinetic studies of curcumin from C-SLNs.     

Carbon determination in human teeth by activation with He-3 ions

A nuclear analytical method, involving activation with³He ions, was developed to determine carbon content in human teeth with well-documented histories. The tooth samples were irradiated with 2.7-MeV³He particles at 50 nA intensity, and the activity of¹⁴O induced through the reaction¹²C(³He, n)¹⁴O, determined by counting the 2.31-MeV gammas. Different dental hard tissues were studied separately. A solid piece of silver steel, the carbon content of which was accurately determined by chemical means, was used as the standard. The carbon content in different teeth varied from 4–7%. The overall experimental accuracy was better than 4.5%.

     

Identification and quantification of five macrolide antibiotics in several tissues, eggs and milk by liquid chromatography–electrospray tandem mass spectrometry

We present an electrospray high-performance liquid chromatographic tandem mass spectrometric (HPLC–MS–MS) method capable of determining in several tissues (muscle, kidney, liver), eggs and milk the following five macrolides: tylosin, tilmicosin, spiramycin, josamycin, erythromycin. Roxithromycin was used as an internal standard. The method uses extraction in a Tris buffer at pH 10.5, followed by protein precipitation with sodium tungstate and clean-up on an Oasis solid-phase extraction column. The HPLC separation was performed on a Purospher C₁₈ column (125×3 mm I.D.) protected by a guard column, with a gradient of aqueous 0.1 M ammonium acetate–acetonitrile as the mobile phase at a flow-rate of 0.7 ml min⁻¹. Protonated molecules served as precursor ions for electrospray ionisation in the positive ion mode and four product ions were chosen for each analyte for multiple reaction monitoring (MRM). A validation study was conducted to confirm the five macrolides by MRM HPLC–MS–MS analysis of a negative control and fortified samples. All of the samples analysed were confirmed with four ions. The ion ratio reproducibility limit ranged from 2.4 to 15%. All compounds could be detected and quantified at half-maximum residue limits (MRLs). The method is specific, quantitative and reproducible enough to conform to European Union recommendations within the concentration range 0.5 MRL–2 MRL (accuracy: 80 to 110%, relative standard deviation: 2 to 13%). This whole method allows extraction and analysis of up to 50 samples per day.     

Application of a fluoride selective electrode to the stoichiometric analysis of lithium carbonate for nuclear purposes

An improved version of a potentiometric method for the determination of lithium has been applied to the accurate, high precision analysis of lithium carbonate, a raw material for the solid-state synthesis of lithium metazirconate (Li₂ZrO₃). This is one of the most promising tritium breeders in fusion reactors. The analytical procedure consisted of a decomposition of the sample in hydrochloric acid followed by evaporation to dryness. The residual lithium chloride was then dissolved in ethanol and titrated with ammonium fluoride in water-ethanol (1 + 1). A lanthanum fluoride solid-state ion-selective electrode was used as end point detector. By increasing the analyte concentration and by dissolving the titrant in the mixed solvent, more accurate results were achieved with respect to the older procedure. A comparison of the results obtained by this method and those obtained by ion-exchange showed no significant difference (P = 0.05). The precision in terms of relative standard deviation was 0.3-0.4%.     

A novel method to measure self-association of small amphipathic molecules: temperature profiling in reversed-phase chromatography

Biophysical techniques such as size-exclusion chromatography, sedimentation equilibrium analytical ultracentrifugation, and non-denaturing gel electrophoresis are the classical methods for determining the self-association of molecules into dimers, trimers, or other higher order species. However, these techniques usually require high (mg/ml) loading concentrations to detect self-association and also possess a lower size limit that is dependent on the ability of the technique to resolve monomeric from higher order species. Here we describe a novel, sensitive method with no upper or lower molecular size limits that indicates self-association of molecules driven together by the hydrophobic effect under aqueous conditions. "Temperature profiling in reversed-phase chromatography" analyzes the retention behavior of a sample over the temperature range of 5-80 degrees C during gradient elution reversed-phase high-performance liquid chromatography. Because this technique greatly increases the effective concentration of analyte upon adsorption to the column, it is extremely sensitive, requiring very small sample quantities (microgram or less). In contrast, the classical techniques mentioned above decrease the effective analyte concentration during analysis, decreasing sensitivity by requiring larger amounts of analyte to detect molecular self-association. We demonstrate the utility of this technique with 14-residue cyclic and linear cationic peptides (<2000 Da) based on the sequence of the de novo-designed cytolytic peptide, GS14. The only requirements for the analyte molecule when using this technique are its ability to be retained on the reversed-phase column and to be subsequently removed from the column during gradient elution.     

Determination of nitrate in blood by gas chromatography and gas chromatography–mass spectrometry

We devised a sensitive and simple method for determining nitrate in whole blood, using an extractive alkylation technique. Nitrate in whole blood was reduced to nitrite by hydrazine sulfate in the presence of Cu²⁺ and Zn²⁺ as catalysts, and alkylated with pentafluorobenzyl bromide using tetradecyldimethylbenzylammonium chloride as the phase-transfer catalyst. The obtained derivative was analyzed qualitatively by gas chromatography–mass spectrometry and quantitatively by gas chromatography with electron-capture detection. The detection limit of nitrate in whole blood was 0.01 mM. The calibration curve was linear over the concentration range from 0.02 to 1.0 mM for nitrate in whole blood. The accuracy and precision of the method were evaluated and the relative standard deviations were found to be within 10%. Using this method, the blood nitrate levels of two victims who committed suicide by inhaling automobile exhaust gas were determined.     

Biological monitoring of dinitrotoluene by gas chromatographic–mass spectrometric analysis of 2,4-dinitrobenzoic acid in human urine

The method of analysis described permits the determination of 2,4-dinitrobenzoic acid down to the lower μg l⁻¹ range in the urine of persons exposed to dinitrotoluene. 2,4-Dinitrobenzoic acid is the main metabolite of 2,4-dinitrotoluene and technical dinitrotoluene. After acidic hydrolysis, which served to release the conjugated part of the 2,4-dinitrobenzoic acid, the analyte was selectively separated from the urine matrix via various extraction steps and then derivatised to the methyl ester. Quantitative analysis was carried out using capillary gas chromatography and mass selective detection. 3,5-Dinitrobenzoic acid was used as an internal standard. The detection limit was 1 μg l⁻¹ urine. The relative standard deviations of within-series imprecision were between 5 and 6%. The relative recoveries were between 91 and 110% depending on the concentration. The analytical method developed as part of this study was used to investigate a collective consisting of 82 urine samples from persons working in the area of explosives disposal. The concentrations of 2,4-dinitrobenzoic acid determined ranged from the detection limit to 95 μg l⁻¹ urine. The method allowed the quantification of low-level internal exposure to dinitrotoluene.