Reference High-performance Liquid Chromatography Method Research Articles

Water-Soluble Photoluminescent Adenosine-Functionalized Gold Nanoclusters as Highly Sensitive and Selective Receptors for Riboflavin Detection in Rat Brain.

Simple, selective, and sensitive detection of cerebral riboflavin is of great significance due to the vital roles of riboflavin in physiological and pathological processes. In the work, water-soluble photoluminescent adenosine-functionalized gold nanoclusters (Ade-AuNCs) are exploited as highly sensitive and selective receptors for cerebral riboflavin detection. The Ade-AuNCs are prepared under aqueous conditions by the one-step "synthesis-functionalization integration" strategy, using chloroauric acid as gold precursors and adenosine as outer-shell ligands. During the Ade-AuNCs synthesis process, adenosine and ascorbic acid are demonstrated to respectively serve as a stabilizer and a reductant, and citrate buffer plays multiple roles including a pH regulator, reductant, and complexing agent. The added riboflavin causes photoluminescence quenching of Ade-AuNCs, and the quenching photoluminescence is applied for well quantifying riboflavin in the range of 0.005-0.1 nM with a detection limit of 0.002 nM. The detailed analytical characterizations reveal that the photoluminescence quenching results from the static photoinduced electron transfer process from the surface functional Ade-AuNCs to riboflavin and the strong affinity between Ade-AuNCs and riboflavin. Moreover, the Ade-AuNC-based sensor exhibits a high selectivity for riboflavin over metal ions, anions, amino acids, and biological substances that possibly exist in the rat brain. Finally, by coupling the microdialysis technique, the proposed sensor is successfully applied to detect riboflavin in living rat brain microdialysates with a basal value of 13.1 ± 2.5 nM (n = 3), and the results are comparable well with those from a reference high-performance liquid chromatography method.

Flow Injection Analysis System Coupled to Chronoamperometry and Boron-Doped Diamond Electrode for Determination of Synthetic Hormones 17α-Ethinylestradiol and Cyproterone Acetate

Flow injection analysis with chronoamperometry detection using a cathodically pretreated boron-doped diamond electrode (CPT-BDDE) was employed to develop and optimize a novel method for the determination of the hormones 17-α-ethinylestradiol (EE2) and cyproterone acetate (CPA). The electrochemical properties of EE2 and CPA were evaluated by cyclic voltammetry and oxidation peaks were observed at 0.97 V and 1.60 V in 0.04 mol L−1 Britton-Robinson buffer (pH 2.0). Analytical curves were linear in the concentration range from 0.070 to 10.0 µmol L−1 for EE2 and 1.0 to 80.0 µmol L−1 for CPA with detection limits of 0.023 and 0.23 µmol L−1, respectively. The developed methods presented good precision and accuracy for EE2 and CPA quantification in commercial pharmaceutical samples. The obtained results were satisfactory and consistent compared with a reference high-performance liquid chromatography method at a confidence level of 95%. Furthermore, recovery studies carried out in synthetic urine and lake water show that these methods do not suffer any significant matrix interferences and are potentially applicable for the determination of these hormones in real samples. Thus, the developed procedure for EE2 and CPA determination are simple and fast, with an analytical frequency of ∼83 determinations per hour, and attractive for routine analysis.

Voltammetric determination of acetaminophen in pharmaceutical preparations and human urine using glassy carbon paste electrode modified with reduced graphene oxide

A completely new direct voltammetric method has been developed for determination of acetaminophen (APAP), known as popular analgesic drug. The present electroanalytical method is based on anodic oxidation of APAP at the glassy carbon paste electrode modified with reduced graphene oxide (RGO). Key experimental conditions were studied, resulting in a set of optimal conditions: acetate buffer (pH 5.0) as working medium electrolyte, content of RGO, parameters of squarewave voltammetry including the potential step of 5 mV, potential amplitude of 50 mV, and frequency of 40 Hz. If peak area is used for evaluation, a linear range from 1.2 × 10–6 to 2.2 × 10–4 mol L−1 characterized by determination coefficient of 0.9971, limits of quantification and detection, 9.3 × 10–7 mol L−1 and 3.1 × 10–7 mol L−1, respectively, will be obtained. Under validation, the precision was described by relative standard deviation of 2.9% for the model sample analysis. Finally, the developed voltammetric method was compared with a reference high-performance liquid chromatography method in the analysis of commercially available pharmaceutical preparation and human urine collected from five healthy volunteers.Graphical abstract

Clinical method evaluation of hemoglobin S and C identification by top-down selected reaction monitoring and electron transfer dissociation

BackgroundBiological diagnosis of hemoglobin disorders is a complex process relying on the combination of several analytical techniques to identify Hb variants in a particular sample. Currently, hematology laboratories usually use high-performance liquid chromatography (HPLC), capillary electrophoresis and gel-based methods to characterize Hb variants. Co-elution and co-migration may represent major issues for precise identification of Hb variants, even for the most common ones such as Hb S and C.MethodsWe adapted a top-down selected reaction monitoring (SRM) electron transfer dissociation (ETD) mass spectrometry (MS) method to fit with a clinical laboratory environment. An automated analytical process with semi-automated data analysis compatible with a clinical practice was developed. A comparative study between a reference HPLC method and the MS assay was performed on 152 patient samples.ResultsThe developed workflow allowed to identify with high specificity and selectivity the most common Hb variants (Hb S and Hb C). Concordance of the MS-based approach with HPLC was 71/71 (100%) for Hb S and 11/11 (100%) for Hb C.ConclusionsThis top-down SRM ETD method can be used in a clinical environment to detect Hb S and Hb C.

In-Process Control Assay of Pharmaceutical Microtablets Using Hyperspectral Imaging Coupled with Multivariate Analysis.

Monitoring the amount of active pharmaceutical ingredient (API) in finished dosage form is important to ensure the content uniformity of the product. In this report, we summarize the development and validation of a hyperspectral imaging (HSI) technique for rapid in-line prediction of the active pharmaceutical ingredient (API) in microtablets with concentrations varying from 60 to 130% API (w/w). The tablet spectra of different API concentrations were collected in-line using an HSI system within the visible/near-infrared (vis/NIR; 400-1000 nm) and short-wave infrared (SWIR; 1100-2500 nm) regions. The ability of the HSI technique to predict the API concentration in the tablet samples was validated against a reference high-performance liquid chromatography (HPLC) method. The prediction efficiency of two different types of multivariate data modeling methods, that is, partial least-squares regression (PLSR) and principle component regression (PCR), were compared. The prediction ability of the regression models was cross-validated against results generated with the reference HPLC method. The results obtained from the PLSR models showed reliable performance for predicting the API concentration in SWIR region. The highest coefficient of determination (R2p) was 0.96, associated with the lowest predicted error and bias of 4.45 and -0.35%, respectively. Furthermore, the concentration-mapped images of PLSR and PCR models were used to visually characterize the API concentration present on the tablet surface. Based on these results, we suggest that HSI measurement combined with multivariate data analysis and chemical imaging could be implemented in the production environment for rapid in-line determination of pharmaceutical product quality.

At-line determination of pharmaceuticals small molecule's blending end point using chemometric modeling combined with Fourier transform near infrared spectroscopy

This article summarizes the development and validation of a Fourier transform near infrared spectroscopy (FT-NIR) method for the rapid at-line prediction of active pharmaceutical ingredient (API) in a powder blend to optimize small molecule formulations. The method was used to determine the blend uniformity end-point for a pharmaceutical solid dosage formulation containing a range of API concentrations. A set of calibration spectra from samples with concentrations ranging from 1% to 15% of API (w/w) were collected at-line from 4000 to 12,500cm−1. The ability of the FT-NIR method to predict API concentration in the blend samples was validated against a reference high performance liquid chromatography (HPLC) method. The prediction efficiency of four different types of multivariate data modeling methods such as partial least-squares 1 (PLS1), partial least-squares 2 (PLS2), principal component regression (PCR) and artificial neural network (ANN), were compared using relevant multivariate figures of merit. The prediction ability of the regression models were cross validated against results generated with the reference HPLC method. PLS1 and ANN showed excellent and superior prediction abilities when compared to PLS2 and PCR. Based upon these results and because of its decreased complexity compared to ANN, PLS1 was selected as the best chemometric method to predict blend uniformity at-line. The FT-NIR measurement and the associated chemometric analysis were implemented in the production environment for rapid at-line determination of the end-point of the small molecule blending operation.

Rapid and simple immunochemical screening combined with hand‐shaking extraction for thiamethoxam residue in agricultural products

With the aim of expanding the applicability of a kit-based enzyme-linked immunosorbent assay (ELISA) for the neonicotinoid insecticide thiamethoxam, the ELISA was newly applied to three kinds of agricultural samples (green pepper, eggplant and spinach). To offer the ELISA as a screening analysis for thiamethoxam residues, a rapid and simple method of extraction by hand-shaking was used, and speed-up and simplification of the sample treatment before the ELISA analysis were examined. Finally, the validity of the ELISA combined with the proposed extraction method was verified against a reference high-performance liquid chromatography (HPLC) method using real-world agricultural samples. There were no marked matrix effects derived from green pepper and eggplant extracts. On the other hand, although the effect due to a pigment in spinach extract on the assay performance was significant, it was effectively avoided by increasing the dilution level of the spinach extract. For thiamethoxam-spiked samples, acceptable recoveries of 97.9-109.1% and coefficients of variation of 0.3-11.5% were obtained. Inspection of the validity of the ELISA by comparison with the reference HPLC method showed that the two analytical results were very similar, and a high correlation was found between them (r>0.997). The evaluated ELISA combined with hand-shaking extraction provided a rapid and simple screening analysis that was quantitative and reliable for the detection of thiamethoxam in complex agricultural products.

Rapid and sensitive electrochemical determination of codeine in pharmaceutical formulations and human urine using a boron-doped diamond film electrode

An unmodified boron-doped diamond film electrode was used for the first time as a sensitive and selective electrochemical sensor for the determination of codeine by the use of differential pulse voltammetry. Codeine provided a single well-defined oxidation peak at +1.0V vs. Ag/AgCl in Britton-Robinson buffer solution at pH 7.0. Using the optimal differential pulse voltammetric conditions (modulation amplitude of 50mV, modulation time of 40ms and scan rate of 50mVs−1), the detection limit of 0.08μM, the linear response of peak current on codeine concentration in the range from 0.1 to 60μM (R2=0.998, n=6) and relative standard deviation of 0.9% at 10μM concentration level (n=10) were achieved without any electrode surface modification. The influence of potential interfering agents on the current response was also studied and the results indicated that the proposed method was sufficiently selective. The method was successfully applied in the determination of codeine in real samples including pharmaceutical tablets and human urine with results similar to those declared by manufacturer and obtained by reference high-performance liquid chromatography method, respectively. The typical benefits of the method may be summarized as: rapidity (20 determinations per hour), sensitivity and selectivity, low cost and elaborateness, simplicity, wide linear concentration range, low detection limit and excellent repeatability. It might also represent the competitive alternative to the existing analytical methods in monitoring of drug abuse.

A competitive immunoassay with a surrogate calibrator curve for aflatoxin M 1 in milk

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M 1 (AFM 1) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM 1 surrogate, was generated by immunizing rabbits with F(ab′) 2 fragments from the anti-AFM 1 monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM 1 mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM 1 and the anti-Id antibody ( y = 31.91 x − 8.47, r = 0.9997). The assay was applied to analyze AFM 1 in spiked milk samples. The IC 50 value of the surrogate calibrator curve was 2.4 μg mL −1, and the inter-assay and intra-assay variations were less than 10.8%; recovery ranged from 85.2 to 110.9%. A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained ( y = 0.81 x + 9.82, r = 0.9922).

Fluorescence-Based High-Throughput Screening Assay for Drug Interactions with UGT1A6

The increasing awareness and the rising importance of UDP-glucuronosyltransferases (UGTs) in the pharmacokinetics of drugs have evoked a need to develop more powerful tools for studying the role of UGTs in the metabolism of drug candidates. To this end, we have developed a fluorescent high-throughput screening assay for screening potential inhibitors and/or substrates for recombinant human UGTs-here, for the UGT1A6. The assay is based on the increase in fluorescence intensity when 1-naphthol is glucuronidated. The formation of the highly fluorescent product, 1-naphthylglucuronide, is followed at excitation wavelengths of 295 and 300 nm with fixed emission (335 nm) in real time directly from the reaction mixture. A probe concentration of 5 μM with 2.5 μg of total protein in phosphate buffer at pH 7.4 with 5% dimethyl sulfoxide resulted in optimal linearity and acceptable signal separation (signal-to-base, 3.0) for the probe reaction. The interactions of test compounds with the enzyme are detected as lower rate of 1-naphthylglucuronide formation and thus lower rate of fluorescence increase. The success of the assay was first demonstrated with the known UGT1A6 substrates 4-hydroxyindole and scopoletin (Z' factor ≥0.5) and later with nonsteroidal anti-inflammatory drugs and salicylate derivatives. Diclofenac, 5-methylsalicylic acid, 5-bromosalicylic acid, 5-chlorosalicylic acid, and 5-fluorosalicylic acid decreased the probe glucuronidation rate at 500 μM by >50%. Further, the results gained with the high-throughput screening assay correlated well with the results obtained, in parallel, with the reference high-performance liquid chromatography method.

Pregnancy and Variations of Carbohydrate-Deficient Transferrin Levels Measured by the Candidate Reference HPLC Method

Contrasting data are available on the diagnostic accuracy of carbohydrate-deficient transferrin (CDT) during pregnancy. These differences may depend in part on how CDT was evaluated and expressed. Here, we report on variations of CDT levels in pregnant women using the high performance liquid chromatography (HPLC) candidate reference method. Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, mean corpuscular volume, serum transferrin, urine and serum ethyl glucuronide and CDT were measured in 64 women, self-reporting as non-alcohol abusers (age: median 34, IQR: 28-38), at different stages of normal pregnancy (gestational weeks: median 28, IQR: 8-33). CDT was expressed as percentage of disialotransferrin to total transferrin (%CDT). Transferrin was associated with both %CDT (r=0.66; P<0.001) and gestational week (r=0.68; P<0.001). Interestingly, %CDT was highly correlated with gestational week (r=0.77; P<0.001), even after controlling for the effect of transferrin. Moreover, statistically significant differences in %CDT were also evident between women grouped for pregnancy trimester (first trimester: mean 1.01% (SD 0.19); second trimester: 1.30% (SD 0.14); third trimester: 1.53% (SD 0.22); ANOVA P<0.001). Trend analysis confirmed a proportional increase of %CDT along with pregnancy trimesters (P<0.001). %CDT, measured with the HPLC candidate reference method, is independently associated with gestational week. Differently from what has been previously reported or expected, the relationship between pregnancy and CDT could be more complex. The diagnostic accuracy of CDT for detecting alcohol abuse in a legal context may be limited in pregnant women and the effect of gestational age should be considered.

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M 1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M 1 (AFM 1), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM 1 of 1.74 × 10 9 L/mol and no cross-reactivity to aflatoxin B 1, B 2, G 1 and G 2. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM 1 in milk and infant milk products. Assays were performed in the AFM 1-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.

Development and Validation of an Ultrasensitive Chemiluminescent Enzyme Immunoassay for Aflatoxin M1 in Milk

A fast and ultrasensitive chemiluminescent enzyme immunoassay for aflatoxin M(1) in milk samples has been developed and validated. The method is an indirect competitive type format involving the immobilization of an aflatoxin M(1)-bovine serum albumin conjugate on 384 well black polystyrene microtiter plates and the use of a secondary antibody labeled with horseradish peroxidase detected with a luminol-based substrate. Aflatoxin M(1) standard solutions were prepared in milk-based buffer, and milk samples were analyzed without any cleanup procedure. The limit of quantification was 1 ppt, the coefficient of variation was below 9% for both intra- and interassay precision, and the recovery ranged from 96 to 122%. The method is specific, and other aflatoxins do not significantly cross-react with the antibody. Twenty-four milk samples were analyzed, and a good correlation was observed (y = 0.98x + 1.71, r(2) = 0.98, n = 24) when the data were compared with a reference high-performance liquid chromatography method with a fluorescent detector. The developed method is suitable for an accurate, sensitive, and high-throughput screening of aflatoxin M(1) in milk samples with a reduction of costs and increased detectability, as compared with previously developed immunoassays.