Internal standard in flow injection analysis with amperometric detection

In this work, we describe for the first time the use of the internal standard method in flow injection analysis (FIA) with amperometric detection. The method is based on the application of sequential potential pulses to the working electrode in an electrochemical flow cell. The sequence of potential pulses is selected in such a way that the analyte and internal standard compound are detected and monitored individually and independently at the same working electrode. This approach compensates for random errors associated with variations of flow rate, injection volume, ionic strength difference between standards and samples, and accidental insertion or formation of air bubbles in the carrier stream. In addition, this method can overcome the major drawback of amperometric detection using solid electrodes, which is gradual electrode passivation. To illustrate the potential of this method, the flow-injection amperometric detection of uric acid using [Fe(CN)₆]^3? as an internal standard (IS) is presented as an example.     

Simple and Fast Determination of Warfarin in Pharmaceutical Samples Using Boron‐doped Diamond Electrode in BIA and FIA Systems with Multiple Pulse Amperometric Detection

This paper proposes the use of the boron‐doped diamond electrode (BDDE) in flow and batch injection analysis (FIA and BIA) systems with multiple‐pulse amperometric (MPA) detection for the determination of warfarin (WA) in pharmaceutical formulations. The electrochemical behavior of WA obtained by cyclic voltammetry (CV) in 0.1 mol L⁻¹ phosphate buffer shows an irreversible oxidation process at +1.0 V (vs Ag/AgCl). The MPA was based on the application of two sequential potential pulses as a function of time on BDDE: (1) for WA detection at +1.2 V/100 ms and; (2) for electrode surface cleaning at −0.2 V/200 ms. Both hydrodynamic systems (FIA‐MPA and BIA‐MPA) used for WA determination achieved high precision (with relative standard deviations around 2 %, n =10), wide linear range (2.0−400.0 μmol L⁻¹), low limits of detection (0.5 μmol L⁻¹) and good analytical frequency (94 h⁻¹ for FIA and 130 h⁻¹ for BIA). The WA determination made by the proposed methods was compared to the official spectrophotometric method. The FIA‐MPA and BIA‐MPA methods are simple and fast, being an attractive option for WA routine analysis in pharmaceutical industries.     

Batch Injection Analysis‐Multiple Pulse Amperometric Fingerprint: A Simple Approach for Fast On‐site Screening of Drugs

In this work, the association of batch injection analysis with multiple pulse amperometric detection (BIA‐MPA) is presented as a new approach to obtain drugs fingerprints. To illustrate the potential of this screening method, tablets containing sildenafil as the active substance were used. Here, a sequence of three potential pulses as a function of time (+1.3, +1.6 and +2.1 V) were applied on a boron‐doped diamond electrode while reproducible injections were performed in a BIA cell (wall jet configuration). The chemical profile of the respective drug combined three ratios among the peak currents obtained in each amperogram: R₁=ipa_1.6V/ipa_1.3V, R₂=ipa_2.1V/ipa_1.6V, R₃=ipa_2.1V/ipa_1.3V. This simple protocol allowed discrimination between Viagra® (reference)/generic and two smuggled tablets, as well as pure Viagra® from Viagra® adulterated with other electroactive compounds (caffeine, dipyrone, paracetamol and tadalafil). For comparison, screening of these samples was also performed using square wave voltammetry combined with a chemometric method (principal component analysis), in which was achieved similar discrimination by one or other strategy for the most of drugs. This new BIA‐MPA fingerprinting combines desirable features in forensic science such as low cost, simplicity, high sample throughput (two drugs discerned in less than 30 s) and portability (screening at the place of the seizure).     

On‐Site Determination of Carbendazim,Cathecol and Hydroquinone in Tap Water Using a Homemade Batch Injection Analysis Cell for Screen Printed Electrodes

In this work, we present a simple homemade batch‐injection analysis cell for screen‐printed electrodes (BIA‐SPE). The potential of the proposed system for on‐site analysis was demonstrated by the determination of carbendazim, catechol, and hydroquinone in tap water. The system provided reduced injection volume (30 µL), high analytical frequency (≈200 h^?1) and low detection limits (nanomolar level). Moreover, the BIA‐SPE cell presented better stability (RSD≈0.4 %) than a conventional flow injection cell for SPE (RSD≈5.0 %) in organic media. The proposed homemade BIA‐SPE cell is very simple, inexpensive and can be easily constructed in any laboratory.     

Square‐wave Voltammetric Determination of Propyphenazone,Paracetamol, and Caffeine: Comparative Study between Batch Injection Analysis and Conventional Electrochemical Systems

In this work, the performance of two methods for simultaneous determination of propyphenazone (PRO), paracetamol (PAR), and caffeine (CAF) were compared. One is based on the use of a conventional electrochemical cell (steady‐state condition) and the other on the use of a batch injection analysis (BIA) cell; both systems were associated with square‐wave voltammetric detection (SWV). Three well separated (▵E ≥ 0.25 V) oxidation peaks were obtained for PRO, PAR, and CAF using 0.1 mol L⁻¹ H₂SO₄ as electrolyte and a boron‐doped diamond (BDD) as working electrode. In addition, the electrochemical oxidation mechanism of PRO is being proposed for the first time. The average relative standard deviations of CB and BIA methods were 4.1 % and 3.1 %, respectively. The conventional system presented better limits of detection and the BIA system a significantly greater throughput (four times faster). Statistical comparison between the results obtained with both proposed methods and those obtained by chromatography was carried out and no significant differences were observed (95 % confidence level).     

Electroanalytical Detection of the Pesticide Paraquat by Batch Injection Analysis

Abstract

The Batch‐Injection Analysis (BIA) technique has been applied to the electroanalytical detection of the herbicide paraquat by square wave voltammetry (SWV) during sample injection. The results obtained showed that the herbicide can be detected at µg l^?1 levels with small injection volumes (<100 µl). The time of each measurement was less than two seconds. The BIA method presents many advantages such as being extremely fast, with high reproducibility, good sensitivity and simple without pre‐addition of or changing the supporting electrolyte.     

Use of the internal standardization for difficult sampling by graphite furnace atomic absorption spectrometry

This work shows the potentiality of As as internal standard to compensate errors from sampling of sparkling drinking water samples in the determination of selenium by graphite furnace atomic absorption spectrometry. The mixture Pd(NO₃)₂/Mg(NO₃)₂ was used as chemical modifier. All samples and reference solutions were automatically spiked with 500 μg l⁻¹ As and 0.2% (v/v) HNO₃ by the autosampler, eliminating the need for manual dilutions. For 10 μl dispensed sample into the graphite tube, a good correlation (r=0.9996) was obtained between the ratio of analyte absorbance by the internal standard absorbance and the analyte concentrations. The relative standard deviations (R.S.D.) of measurements varied from 0.05 to 2% and from 1.9 to 5% (n=12) with and without internal standardization, respectively. The limit of detection (LD) based on integrated absorbance was 3.0 μg l⁻¹ Se. Recoveries in the 94-109% range for Se spiked samples were obtained. Internal standardization (IS) improved the repeatability of measurements and increased the lifetime of the graphite tube in ca. 15%.     

Calibration lines passing through the origin with errors in both axes

Linear regression of calibration lines passing through the origin was investigated for three models of y-direction random errors: normally distributed errors with an invariable standard deviation (SD) and log normally and normally distributed errors with an invariable relative standard deviation (RSD). The weighted (weighting factor is x ² _i ), geometric and arithmetic means of the ratios y _i /x _i estimate the calibration slope for these models, respectively. Regression of the calibration lines with errors in both directions was also studied. The x-direction errors were assumed to be normally distributed random errors with either an invariable SD or invariable RSD, both combined with a constant relative systematic error. The random errors disperse the true, unknown x-values about the plotted, demanded x-values, which are shifted by the constant relative systematic error. The systematic error biases the slope estimate while the random errors do not. They only increase automatically the slope estimate uncertainty, in which the uncertainty component reflecting the range of the possible values of the systematic error must be additionally included. Received: 9 May 2000 Accepted: 7 March 2001     

Fast and Selective Simultaneous Determination of Acetaminophen,Aspirin and Caffeine in Pharmaceutical Products by Batch Injection Analysis with Multiple Pulse Amperometric Detection

In this work is presented a method for simultaneous determination of paracetamol (PA), acetylsalicylic acid (ASA) and caffeine (CA) in pharmaceutical tablets, using a bare boron‐doped diamond working electrode (BDDE) coupled to batch injection analysis system with multiple pulse amperometric detection (BIA‐MPA). The optimized sequence of fast potential pulses were applied on BDDE for acquisition of independent amperograms: +1.0 V for PA oxidation, +1.3 V for oxidation of PA and salicylic acid (SA) generated from a previous alkaline hydrolysis of ASA and +1.6 V in which the three analytes are oxidized (PA, SA and CA). Selective determination of PA is performed using the currents obtained at +1.0 V, while SA and CA signals are indirectly obtained using simple subtraction operations between peak currents from each amperogram and correction factors (CF's). The limitations of such approach on the precision and accuracy as function of BIA‐MPA conditions are discussed. Simultaneous determination of the target drugs in pharmaceutical tablets was performed by BIA‐MPA and the results compared to a HPLC‐DAD method. Under optimized conditions, the proposed method exhibits fast responses (180 injections per hour for the simultaneous determination of the three analytes) and suitable precision (RSD_PA: 0.78 %; RSD_SA: 1.09 %; RSD_CA: 2.73 %). BIA‐MPA method is simple, portable and presents relative low‐cost.     

Batch Injection Analysis Utilizing Modified Electrodes with Tetraruthenated Porphyrin Films for Acetaminophen Quantification

Batch injection analysis (BIA) utilizing amperometric detection with glassy carbon electrodes modified with [Co(TPyP){Ru(bipy)₂Cl}₄](TFMS)₅?H₂O porphyrin films were explored for acetaminophen analysis in pharmaceutical formulations. BIA is an interesting alternative for application of electrodes modified with tetraruthenated porphyrins. This sensor exhibited sharp current response peaks, rapid washout and excellent reproducibility for BIA‐amperometric quantification of acetaminophen. Also, a wide linear working range (10^?4 to 10^?6 mol L^?1) as well as high sensitivity and sampling frequency rate (detection limit=1.1×10^?7 mol L^?1, sampling frequency=120 injections/h) and a small volume of analysis (100 μL/injection) was achieved. Furthermore, the proposed method permits the direct quantification of acetaminophen in many pharmaceutical products, avoiding cumbersome processes as previous separations, solvent extraction or sample filtration. The new procedure was applied to the analysis of commercial pharmaceutical products and the results were in excellent agreement with the ones obtained by spectrophotometric method. Accordingly, this amperometric method showed to be very well suited for quality control analysis and other applications with similar requirements.     

Simultaneous determination of vitexin and isovitexin in rat plasma after oral administration of Santalum album L. leaves extract by liquid chromatography tandem mass spectrometry

An LC‐MS/MS method was developed for the simultaneous determination of vitexin and isovitexin in rat plasma, using puerarin as the internal standard (IS). Plasma samples extracted with protein precipitation procedure were separated on a Diamonsil® C₁₈ column (150 × 4.6 mm, 5 µm) with a mobile phase composed of methanol and 0.1% formic acid (45:55, v/v). The detection was accomplished by multiple reaction monitoring mode in positive electrospray ionization source. The optimized mass transition ion‐pairs for quantitation were m/z 431.2 → 311.1 for vitexin and isovitexin, and m/z 415.1 → 295.1 for IS. The total run time was 7.5 min for each injection. The calibration curves were linear (r² > 0.99) over the investigated concentration range (2.00–2000 ng/mL) and the lower limits of quantification were 2.00 ng/mL in rat plasma sample. The intra‐ and inter‐day relative standard deviations were no more than 14.9% and the relative errors were within the range of ?3.2–2.1%. The extraction recoveries for both compounds were between 89.3 and 97.3%. The robust LC‐MS/MS method was further applied in the pharmacokinetic study in Sprague–Dawley rats after oral administration of Santalum album L. leaves extract at a dose of 116 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of 9‐dehydro‐17‐hydro‐andrographolide and sodium 9‐dehydro‐17‐hydro‐andrographolide‐19‐yl sulfate in rat plasma by UHPLC‐ESI‐MS/MS after administration of xiyanping injection: application to a pharmacokinetic study

9‐Dehydro‐17‐hydro‐andrographolide (DHA) and sodium 9‐dehydro‐17‐hydro‐andrographolide‐19‐yl sulfate (DHAS) are active ingredients of xiyanping injection in clinical use. A simple, rapid and sensitive UHPLC‐ESI‐MS/MS method was developed for the determination of DHA and DHAS in rat plasma, and the pharmacokinetics of DHA and DHAS after intravenous administration of xiyanping injection was investigated. The plasma samples were treated with methanol to precipitate out protein, and the separation of DHA and DHAS was achieved on a Waters BEH C₁₈ column with a mobile phase consisting of acetonitrile and 10 mmol/L ammonium acetate solution at a flow rate of 0.4 mL/min. DHA, DHAS and the internal standard (internal standard, IS) diethylstilbestrol were detected at negative ion mode. The precursor‐product ion pairs used in multiple reaction monitoring mode were: m/z 349.1 → 286.9 (DHA), m/z 428.9 → 96.0 (DHAS) and m/z 267.1 → 236.9 (IS). Calibration curves offered satisfactory linearity within the test range, and all correlation coefficients were >0.995. The lower limit of detection of DHA and DHAS in plasma samples were determined to be 0.1 ng/mL. The lower limit of quantitation was 0.5 ng/mL for DHA and DHAS. All the recoveries of the quality control samples were in the range of 86.0–102.4%. The ratios of matrix effect were between 89.2 and 105.1%. The method was fully validated and successfully applied to the pharmacokinetic study of DHA and DHAS in rats. The study showed that both DHA and DHAS were distributed and eliminated rapidly in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

A liquid chromatography–mass spectrometric method for the quantification of azithromycin in human plasma

A liquid chromatographic mass spectrometric assay for the quantification of azithromycin in human plasma was developed. Azithromycin and imipramine (as internal standard, IS) were extracted from 0.5 mL human plasma using extraction with diethyl ether under alkaline conditions. Chromatographic separation of drug and IS was performed using a C₁₈ column at room temperature. A mobile phase consisting of methanol, water, ammonium hydroxide and ammonium acetate was pumped at 0.2 mL/min. The mass spectrometer was operated in positive ion mode and selected ion recording acquisition mode. The ions utilized for quantification of azithromycin and IS were m/z 749.6 (M + H) ⁺ and m/z 591.4 (fragment) for azithromycin, and 281.1 m/z for internal standard; retention times were 6.9 and 3.4 min, respectively. The calibration curves were linear (r² > 0.999) in the concentration ranges of 10–1000 ng/mL. The mean absolute recoveries for 50 and 500 ng/mL azithromycin and 1 µg/ mL IS were >75%. The percentage coefficient of variation and mean error were <11%. Based on validation data, the lower limit of quantification was 10 ng/mL. The present method was successfully applied to determine azithromycin pharmacokinetic parameters in two obese volunteers. The assay had applicability for use in pharmacokinetic studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Suitability of a fully 13C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up

Very often, the accuracy of quantitative analytical methods for the determination of mycotoxins by liquid chromatography (LC)-mass spectrometry (MS) and LC-MS/MS is limited by matrix effects during the ionization process in the MS source. Stable isotope labeled standards are best suited to correct for matrix effects and to improve both the trueness and the precision of analytical methods employing LC-MS and LC-MS/MS. This paper describes the successful use of fully ¹³C isotope labeled deoxynivalenol [(¹³C₁₅)DON] as an internal standard (IS) for the accurate determination of DON in maize and wheat by LC electrospray ionization MS/MS. To show the full potential of (¹³C₁₅)DON as IS, maize and wheat extracts were analyzed without further cleanup. Subsequent to calibration for the LC-MS end determination, DON was quantified in matrix reference materials (wheat and maize). Without consideration of the IS, apparent recoveries of DON were 29±6% (n=7) for wheat and 37±5% (n=7) for maize. However, the determination of DON in the reference materials yielded 95±3% (wheat) and 99±3% (maize) when (¹³C₁₅)DON was used as an IS for data evaluation.     

New Determination Scheme of p‐Aminophenol by MnO2 Modified Electrode Coupled with Flow Injection Analysis

New determination scheme of p‐aminophenol by using MnO₂ as a preoxidant is demonstrated in this work. In the flow injection system, the p‐aminophenol is oxidized to quinoneimine by MnO₂ at up‐stream, which can be detected at a suitable reductive potential. After optimization, the linear range of PAP is started from 1 μM to 30 μM (R²=0.999), the estimated detection limit (S/N=3) is 0.28 μM. Two real samples are studied and excellent recoveries are achieved by using standard addition method.     

Simultaneous determination of colchicine and febuxostat in rat plasma: Application in a rat pharmacokinetic study

A selective, sensitive and rapid LC–MS/MS method has been developed and validated as per US Food and Drug Administration regulatory guidelines for the simultaneous quantitation of colchicine and febuxostat in rat plasma. Colchicine and febuxostat were extracted from the rat plasma using 10% tert-butyl methyl ether in ethyl acetate using colchicine-d₆ as an internal standard (IS). The chromatographic separation of colchicine, febuxostat and the IS was achieved using a mobile phase comprising 5 mm ammonium formate and 0.025% formic acid in acetonitrile (20:80, v/v) in isocratic mode on an Eclipse XDB-C₁₈ column. The injection volume and flow rate were 5.0 μl and 0.9 ml/min, respectively. Colchicine and febuxostat were detected by positive electrospray ionization in multiple reaction monitoring mode using transition pairs (Q1 → Q3) of m/z 400.10 → 358.10 and 317.05 → 261.00, respectively. The assay was linear in the ranges of 0.25–254 and 2.60–622 ng/ml for colchicine and febuxostat, respectively. The inter- and intra-day precision values were 0.58–13.0 and 1.03–4.88% for colchicine and febuxostat, respectively. No matrix or carryover effects were observed during the validation. Both analytes were stable on the bench-top, in the autosampler and in storage (freeze–thaw cycles and long-term storage at −80 ° C). A pharmacokinetic study in rats was performed to show the applicability of the validated method.     

A highly sensitive and efficient UPLC‐MS/MS assay for rapid analysis of tedizolid (a novel oxazolidinone antibiotic) in plasma sample

Tedizolid (TDZ) is a novel oxazolidinone class antibiotic, indicated for the treatment of acute bacterial skin and skin structure infections in adults. In this study a highly sensitive UPLC‐MS/MS assay was developed and validated for the determination of TDZ in rat plasma using rivaroxaban as an internal standard (IS). Both TDZ and IS were separated on an Acquity UPLC BEH? C₁₈ column using an isocratic mobile phase comprising of acetonitrile–20 mm ammonium acetate (85:15, v/v), eluted at 0.3 mL/min flow rate. The plasma sample was processed by liquid liquid extraction technique using ethyl acetate as an extracting agent. The analyte and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.09 > 343.10 for TDZ and m/z 435.97 > 144.94 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 0.74–1500 ng/mL and the lower limit of quantification was 0.74 ng/mL only. The developed assay was validated following standard guidelines for bioanalytical method validation (US Food and Drug Administration) and all the validation results were within the acceptable limits. The developed assay was successfully applied into a pharmacokinetic study in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous Flow Injection Analysis of Paracetamol and Ascorbic Acid with Multiple Pulse Amperometric Detection

The present work reports a simple and quick strategy for simultaneous determination of paracetamol (PC) and ascorbic acid (AA) in pharmaceutical formulations using flow injection method with multiple pulse amperometric detection. The method allows the resolution of the mixture without chemical pretreatment of the sample or electrode modification or the use of chemometric techniques for data analysis. The compounds are detected by applying four sequential pulses (waveform) in function of time to a three‐electrode amperometric system that uses a wall‐jet cell with gold as working electrode. AA is direct detected at +0.40 V and PC is indirectly detected at 0.0 V by the reduction (desorption) of the oxidation product (N‐acetyl‐p‐benzoquinoneimine) electrochemically generated at +0.65 V. The fourth potential pulse (?0.05 V) is applied for the complete regeneration (cleaning) of the gold electrode surface. The linear response range was optimized between 5 and 24 mg L^?1 for AA and 50 and 240 mg L^?1 for PC. The difference between the two responses ranges (10‐fold) present correlation with the concentration of these compounds in two different pharmaceutical formulations available in the Brazilian market. The analytical frequency was calculated in 60 injections per hour. The use of the proposed methodology for PC quantification in the presence of higher AA concentrations was also carried out. Using the standard addition method, it was possible to detect PC in trace levels (LD=0.2 mg L^?1) in the presence of 880‐fold more of AA (176 mg L^?1).     

Solenoid Micro‐pumps: A New Tool for Sample Introduction in Batch Injection Analysis Systems with Electrochemical Detection

This work presents the use of solenoid micro‐pumps as a new strategy for sample introduction in batch‐injection analysis (BIA). The volume of solution dispensed on each pulse of the solenoid micro‐pump (μL) is used as fixed and reproducible injection volume for BIA. In this system, the injection steps are possible in stopped flow mode resulting in low background noise levels, which would not be possible under continuous flow conditions and using solenoid micro‐pumps. As a proof‐of‐concept, amperometric and square‐wave voltammetric (SWV) determination of dopamine was demonstrated as well as anodic‐stripping voltammetry (ASV) of metals. The micro‐pump provided injections of 14 μL of solution per pulse at 512 μL s⁻¹ over the electrode during electrochemical measurement. Moreover, fast injections of analyte or electrolyte were programmed during deposition or conditioning steps of ASV for analyte preconcentration or electrode cleaning. The proposed system improved limits of detection and sensitivity (2‐fold), precision and sample throughput in comparison with traditional BIA due to enhanced mass transfer and consequent reduced dispersion of analyte, and possible control of injections without analyst intervention. This work opens new possibilities of applications of the BIA system, including on‐line sample treatment (derivatization or dilution steps).     

Ultra‐high‐performance liquid chromatography tandem mass spectrometry method for the determination of gambogenic acid in dog plasma and its application to a pharmacokinetic study

A highly sensitive and rapid ultra‐high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of gambogenic acid in dog plasma. Gambogic acid was used as an internal standard (IS). After a simple liquid–liquid extraction by ethyl acetate, the analyte and internal standard were separated on an Acquity BEH C₁₈ (100 × 2.1 mm, 1.7 µm; Waters ) column at a flow rate of 0.2 mL/min, using 0.1% formic acid–methanol (10:90, v/v) as mobile phase. Electrospray ionization source was applied and operated in the positive ion mode. Multiple reaction monitoring mode with the transitions m/z 631.3 → 507.3 and m/z 629.1 → 573.2 was used to quantify gambogenic acid and the internal standard, respectively. The calibration curves were linear in the range of 5–1000 ng/mL, with a coefficient of determination (r) of 0.999 and good calculated accuracy and precision. The low limit of quantification was 5 ng/mL. The intra‐and inter‐day precisions (relative standard deviations) were <15%. The methodology recoveries were more than 66.63%. This validated method was successfully applied to a pharmacokinetic study after intravenous injection administration of gambogenic acid in dogs at a dose of 1 mg/kg. Copyright © 2014 John Wiley & Sons, Ltd.