Determination of amoxicillin in pharmaceuticals using sequential injection analysis (SIA): Evaluation of the presence of interferents using multivariate curve resolution

In this study, we report a method for determining amoxicillin in pharmaceuticals using sequential injection analysis (SIA) with a diode-array spectrophotometric detector. Before determining the amoxicillin, we use multivariate curve resolution with alternating least squares (MCR-ALS) to investigate whether any interferents are present. With a suitable analytical sequence, we can use SIA to generate a pH gradient and, for each sample, obtain a matrix of data that have been analysed by several chemometric techniques based on multivariate analysis: principal components analysis (PCA), simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) and MCR-ALS. In this way we obtain the concentration profiles and spectra of the species in the sample.We studied six pharmaceuticals containing amoxicillin. Two of these pharmaceuticals contained no interferents, one contained an interferent but amoxicillin had a selective spectral area with respect to it, and the other three contained interferents. For the first three samples, we set up a system of univariate calibration, which determines amoxicillin quickly (it can analyse 20 samples/h) using inexpensive instrumentation and reactives.     

Sequential injection analysis for the simultaneous determination of clavulanic acid and amoxicillin in pharmaceuticals using second-order calibration.

In this paper, we report on a method for quantifying clavulanic acid and amoxicillin simultaneously in pharmaceuticals using sequential injection analysis (SIA) with a diode-array spectrophotometric detector and multivariate curve resolution with alternating least squares (MCR-ALS). We optimized the experimental parameters so that the analytical sequence could distinguish the concentrations and spectrum profiles of the species of interest with optimum resolution quality. After establishing the optimum conditions, we quantified clavulanic acid and amoxicillin in four pharmaceuticals. In most cases our results were slightly higher than those in the prospectus of the pharmaceutical. The relative standard deviations were below 5% for amoxicillin and below 7% for clavulanic acid. These results are acceptable because, to prevent degradation due to bacteriostatic activity, the concentration of the main reactant is usually higher.     

Influence of natural organic matter on the screening of pharmaceuticals in water by using liquid chromatography with full scan mass spectrometry

The influence of natural organic matter on the screening of pharmaceuticals in water was determined by using high resolution liquid chromatography (HRLC) combined with full scan mass spectrometry (MS) techniques like time of flight (ToF) or Orbitrap MS. Water samples containing different amount of natural organic matter (NOM) and residues of a set of 11 pharmaceuticals were analyzed by using Exactive Orbitrap? LC-MS. The samples were screened for residues of pharmaceuticals belonging to different classes like benzimidazoles, macrolides, penicillins, quinolones, sulfonamides, tetracyclines, tranquillizers, non-steroidal anti-inflammatory drugs (NSAIDs), anti-epileptics and lipid regulators. The method characteristics were established over a concentration range of 0.1-500 μg L(-1). The 11 pharmaceuticals were added to two effluent and two influent water samples. The NOM concentration within the samples ranged from 2 to 8 mg L(-1) of dissolved organic carbon. The HRLC-Exactive Orbitrap? LC-MS system was set at a resolution of 50,000 (FWHM) and this selection was found sufficient for the detection of the list of pharmaceuticals. With this resolution setting, accurate mass measurements with errors below 2 ppm were found, despite of the NOM concentration of the different types of water samples. The linearities were acceptable with correlation coefficients greater than 0.95 for 30 of the 51 measured linearities. The limit of detection varies between 0.1 μg L(-1)and 100 μg L(-1). It was demonstrated that sensitivity could be affected by matrix constituents in both directions of signal reduction or enhancement. Finally it was concluded that with direct shoot method used (no sample pretreatment) all compounds, were detected but LODs depend on matrix-analyte-concentration combination. No direct relation was observed between NOM concentration and method characteristics. For accurate quantification the use of internal standards and/or sample clean-up is necessary. The direct shoot method is only applicable for qualitative screening purposes. The use of full scan MS makes it possible to search for unknown contaminants. With the use of adequate software and a database containing more than 50,000 entries a tool is available to search for unknowns.     

Use of multivariate curve resolution for determination of chromium in tanning samples using sequential injection analysis

We report a method for determining total chromium in tanning samples using sequential injection analysis (SIA) with a diode-array spectrophotometric detector. With a suitable analytical sequence CrO₄^2– is converted to Cr₂O₇^2– inside the tubes of the SIA system, after total oxidation of chromium(III). A data matrix is obtained and analysed by several chemometric techniques based on multivariate analysis: principal components analysis, simple-to-use interactive self-modelling mixture analysis, and multivariate curve resolution-alternating least-squares. We studied several samples from different stages of a tanning process. Two of these samples were easily oxidized but the others needed more extreme conditions. The analytical sequence prepared, which was based on obtaining a pH gradient and used H₂SO₄ as reagent, is valid and independent of the level of oxidation needed for the sample. We established a calibration model and evaluated the figures of merit. In some samples we found interferents. With this method the amounts of chromium in each sample were quantified and the results were statistically similar to those obtained by use of the reference method, atomic absorption spectrometry.     

Separation of synthetic food colourants in the mixed micellar system. Application to pharmaceutical analysis

The paper presents a rapid method for the determination of commonly used synthetic food dyes by micellar electrokinetic capillary chromatography. Detection and separation conditions allowing complete resolution of 15 synthetic food colourants were investigated. The effect of different surfactants on the analytes mobility in relation to their structure was tested. After optimization procedure a dual micellar system was selected. All food dyes were separated in less then 20 min using a fused silica capillary in the borate/dodecylsulfate/deoxycholate buffer containing acetonitrile as organic modifier. The detection wavelength was set at 210nm. The method was successfully validated by determination of linearity ranges, detection limits, precision and repeatability for all colourants tested. In order to apply the method for pharmaceutical analysis a sample pretreatment procedures were found. Liquid pharmaceuticals were used as it or just after dilution with water. From tablets or capsules the colourants were isolated by adsorption on acidic aluminium oxide. The method was used for identification and if possible for quantification the synthetic food dyes in pharmaceuticals. The analytes are detectable at a concentration level 0.3-0.8 microg ml(-1).     

Sensitive determination of indomethacin in pharmaceuticals and urine by sequential injection analysis and optosensing

A selective and sensitive method based on coupling of sequential injection analysis (SIA) and optosensing was developed and applied to the determination of indomethacin in pharmaceutical and urinary samples. After alkaline hydrolysis, the fluorescent product generated from indomethacin is inserted in the flow system, transitorily retained on an active solid support (Sephadex QAE A-25) filling the flowcell, and monitored at 283/371 nm (lamda ex / lamda em). The system was calibrated for two sample volumes, 100 and 1000 microL. It showed a linear dynamic range of 0.5-6.5 ng/mL, with an LOD of 0.15 ng/mL and an RSD of 3.9% (n=10) when the highest sample volume was used. The proposed fluorometric SIA optosensor was applied to the determination of indomethacin in both pharmaceuticals and urine samples, and satisfactory results were obtained.     

Sequential injection analysis implementing multiple standard additions for As speciation by liquid chromatography and atomic fluorescence spectrometry (SIA-HPLC-AFS)

An analytical procedure for multiple standard additions of arsenic species using sequential injection analysis (SIA) is proposed for their quantification in seafood extracts. SIA presented flexibility for generating multiple specie standards at the ng mL⁻¹ concentration level by adding different volumes of As(III), As(V), monomethylarsonic (MMA) and dimethylarsinic (DMA) to the sample. The mixed sample plus standard solutions were delivered from SIA to fill the HPLC injection loop. Subsequently, As species were separated by HPLC and analyzed by atomic fluorescence spectrometry (AFS). The proposed system comprised two independently controlled modules, with the HPLC loop acting as the intermediary device. The analytical frequency was enhanced by combining the actions of both modules. While the added sample was flowing through the chromatographic column towards the detection system, the SIA program started performing the standard additions to another sample. The proposed method was applied to spoiled seafood extracts. Detection limits based on 3σ for As(III), As(V), MMA and DMA were 0.023, 0.39, 0.45 and 1.0 ng mL⁻¹, respectively.     

Rapid automated assay of anti-oxidation/radical-scavenging activity of natural substances by sequential injection technique (SIA) using spectrophotometric detection

A PC-controlled sequential injection analysis (SIA) system equipped with a spectrophotometric diode-array detector is used for rapid monitoring and evaluation of antioxidation/radical scavenging activity of biological samples. The automated method is based on the known reaction of stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) with antioxidants in organic or aqueous-organic media resulting in bleaching of DPPH due to its quenching by the interaction with the analytes. The decrease of the absorbance of DPPH (compared to blank experiment carried out with water–ethanol 1:1 instead of the test solution) measured at 525 nm is related to concentration of an antioxidant in the test solution. With the optimised SIA procedure it is possible to detect down to micromolar concentrations of model antioxidants such as ascorbic acid, caffeic acid, (+)-catechin, (–)-epicatechin and rutin and to evaluate the concentration of these antioxidants in the micromolar to millimolar range. The sample throughput is 45 h^–1. Thanks to its rapidity and sensitivity, the proposed SIA method is suitable for performing routine screening tests for the presence of various antioxidants in large series of lyophilised herbal or mushroom extracts (the amount of sample needed for the analysis is several milligrams).     

Factorial design for optimising chromium determination in tanning wastewater

A totally automatized procedure for determining chromium by sequential injection analysis (SIA) linked to multivariate curve resolution with alternating least squares (MCR-ALS) is proposed. With this system Cr(III) is oxidised to chromate (Cr(VI)) and this form is then converted to dichromate in order to obtain second order data. The experimental design method was used to establish the best conditions. The identification of the most influential factors was validated using ANOVA tests. We used this method to successfully analyse total chromium in several aqueous tannery samples from various steps of a tanning process. The results from this method and those from the atomic absorbance spectroscopy (AAS) method are comparable. Sample frequency was 30 samples h^− 1.     

Ultra-short columns and ballistic gradients: considerations for ultra-fast chromatographic liquid chromatographic-tandem mass spectrometric analysis

Ultra-fast chromatographic separations has enabled fast chromatographic method development and rapid analysis for sample quantification. Decreasing over-all analytical time has become a factor of major importance for all aspects of drug discovery. However, merely decreasing chromatographic analysis time by decreasing k' can lead to inconsistent quantitative or qualitative results due to ineffective separations in complex matrices. We have found that by changing column length and gradient slope we can maintain chromatographic integrity of chemically diverse analytes and achieve the analytical speed required for bioanalytical drug discovery quantitative analysis. We have optimized method development strategy by performing separations on 2x20 mm HPLC columns at flow-rates of 1.5 ml/min to 2 ml/min with full linear gradients achieved in 1 min for the quantification of pharmaceuticals and their metabolites from biological matrices. This method development strategy can be readily adapted to other matrices. This paper will discuss the effects of column length and gradient time in ultra-fast chromatographic resolution.     

Sequential injection analysis with dynamic surface tension detection High throughput analysis of the interfacial properties of surface-active samples

A sequential injection analysis (SIA) system is coupled with dynamic surface tension detection (DSTD) for the purpose of studying the interfacial properties of surface-active samples. DSTD is a novel analyzer based upon a growing drop method, utilizing a pressure sensor measurement of drop pressure. The pressure signal depends on the surface tension properties of sample solution drops that grow and detach at the end of a capillary tip. In this work, SIA was used for creating a reagent concentration gradient, and for blending the reagent gradient with a steady-state sample. The sample, consisting of either sodium dodecyl sulfate (SDS) or poly(ethylene glycol) at 1470 g mol⁻¹ (PEG 1470), elutes with a steady-state concentration at the center of the sample plug. Reagents such as Brij®35, tetrabutylammonium (TBA) hydroxide and β-cyclodextrin were introduced as a concentration gradient that begins after the sample plug has reached the steady-state concentration. By blending the reagent concentration gradient with the sample plug using SIA/DSTD, the kinetic surface pressure signal of samples mixed with various reagent concentrations is observed and evaluated in a high throughput fashion. It was found that the SIA/DSTD method consumes lesser reagent and required significantly less analysis time than traditional FIA/DSTD. Four unique chemical systems were studied with regard to how surface activity is influenced, as observed through the surface tension signal: surface activity addition, surface activity reduction due to competition, surface activity enhancement due to ion-pair formation, and surface activity reduction due to bulk phase binding chemistry.     

Development of an automated chemiluminescence flow-through sensor for the determination of 5-aminosalicylic acid in pharmaceuticals: a comparative study between sequential and multicommutated flow techniques

This work is aimed at demonstrating the potential of the implementation of automatic flow systems in optosensors using chemiluminescence detection. With this purpose, two automatic methodologies, multicommutation and sequential injection analysis (SIA), have been applied to the analysis of 5-aminosalicylic acid (ASA). The analyte is determined for the first time making use of its chemiluminescence reaction with permanganate anion, previously immobilized on an appropriate solid support in the detection area. First, the study of the most appropriate commercial flow-through cell and the optimum conditions for the reaction were performed. Second, the main differences in terms of flow variables and analytical parameters for multicommutation and SIA approaches were stated. Both methodologies were applied to the determination of the analyte in pharmaceuticals obtaining satisfactory results. Finally, the advantages and disadvantages of both proposed methods and the recoveries obtained from pharmaceuticals were statistically compared.     

High-resolution mass spectrometry method for the detection, characterization and quantitation of pharmaceuticals in water

The presence of pharmaceuticals in drinking water is an emerging environmental concern. In most environmental testing laboratories, LC-MS/MS assays based on selected reaction monitoring are used as part of a battery of tests used to assure water quality. Although LC-MS/MS continues to be the best tool for detecting pharmaceuticals in water, the combined use of hybrid high-resolution mass spectrometry (HRMS) and ultrahigh pressure liquid chromatography (UHPLC) is starting to become a practical tool to study emerging environmental contaminants. The hybrid LTQ-orbitrap mass spectrometer is suitable for integrated quantitative and qualitative bioanalysis because of the following reasons: (1) the ability to collect full-scan HRMS spectra with scan speeds suitable for UHPLC separations, (2) routine measurement of mass with less than 5 ppm mass accuracy, (3) high mass resolving power, and (4) ability to perform on-the-fly polarity switching in the linear ion trap (LTQ). In the present work, we provide data demonstrating the application of UHPLC-LTQ-orbitrap for the detection, characterization and quantification of pharmaceuticals and their metabolites in drinking water.     

Multiresidue methods for the analysis of pharmaceuticals,personal care products and illicit drugs in surface water and wastewater by solid-phase extraction and ultra performance liquid chromatography–electrospray tandem mass spectrometry

The main aim of the presented research is to introduce a new technique, ultra performance liquid chromatography-positive/negative electrospray tandem mass spectrometry (UPLC-ESI/MS/MS), for the development of new simultaneous multiresidue methods (over 50 compounds). These methods were used for the determination of multiple classes of pharmaceuticals (acidic, basic and neutral compounds: analgesic/anti-inflammatory drugs, antibiotics, antiepileptics, beta-adrenoceptor blocking drugs, lipid regulating agents, etc.), personal care products (sunscreen agents, preservatives, disinfectant/antiseptics) and illicit drugs (amphetamine, cocaine and benzoylecgonine) in surface water and wastewater. The usage of the novel UPLC system with a 1.7 microm particle-packed column allowed for good resolution of analytes with the utilisation of low mobile phase flow rates (0.05-0.07 mL min(-1)) and short retention times (method times of up to 25 min), delivering a fast and cost-effective method. SPE with the usage of Oasis MCX strong cation-exchange mixed-mode polymeric sorbent was chosen for sample clean-up and concentration. The influence of mobile phase composition, matrix-assisted ion suppression in ESI-MS and SPE recovery on the sensitivity of the method was extensively studied. The method limits of quantification were at low nanogram per litre levels and ranged from tenths of ng L(-1) to tens of ng L(-1) in surface water and from single ng L(-1) to a few hundreds of ng L(-1) in the case of wastewater. The instrumental and method intraday and interday repeatabilities were on average less than 5%. The method was successfully applied for the determination of pharmaceuticals in the River Taff (South Wales) and a wastewater treatment plant (WWTP Cilfynydd). Several pharmaceuticals and personal care products were determined in river water at levels ranging from single ng L(-1) to single microg L(-1).     

Factorial design and response surface optimization of spectrophotometric sequential injection analysis of olanzapine formulations

A new spectrophotometric sequential injection analysis (SIA) method for the assay of olanzapine in pharmaceutical formulations was optimized by the factorial design and the response surface approaches. The method was based on the oxidation of olanzapine by an excess amount of permanganate in sulfuric acid media. The reduction of permanganate was spectrophotometrically detected at 570 nm. The 2³ full factorial design was adopted for the optimization of permanganate concentration, sulfuric acid concentration and flow rate. The method was validated based on the IUPAC guidelines. Real pharmaceutical samples were subjected to the proposed SIA method and the results were in satisfactory agreement with those obtained by a previous spectrophotometric method. The full-automation of SIA empowered the proposed method with high repeatability (RSD 1.74%, n = 7) and intermediate-precision (RSD 2.53%, n = 5). Additionally, both automation and miniaturization offered high sampling frequency (26 samples/h). Furthermore, the employment of chemometric optimization enhanced sensitivity of the method with limits of detection and quantification of 1.07 and 3.57 mg/L, respectively. Comparing with previous olanzapine assay methods, which employing conventional analytical techniques, the new SIA method is inexpensive in terms of instrumentation, consumption of reagents and samples as well as effort and manpower. The SIA method is also safer for handling solutions and for the environment.     

Titration of strong and weak acids by sequential injection analysis technique

A sequential injection analysis (SIA) titration method has been developed for acid-base titrations. Strong and weak acids in different concentration ranges have been titrated with a strong base. The method is based on sequential aspiration of an acidic sample zone and only one zone of the base into a carrier stream of distilled water. On their way to the detector, the sample and the reagent zones are partially mixed due to the dispersion and thereby the base is partially neutralised by the acid. The base zone contains the indicator. An LED-spectrophotometer is used as detector. It senses the colour of the unneutralised base and the signal is recorded as a typical SIA peak. The peak area of the unreacted base was found to be proportional to the logarithm of the acid concentration. Calibration curves with good linearity were obtained for a strong acid in the concentration ranges of 10(-4)-10(-2) and 0.1-3 M. Automatic sample dilution was implemented when sulphuric acid at concentration of 6-13 M was titrated. For a weak acid, i.e. acetic acid, a linear calibration curve was obtained in the range of 3x10(-4)-8x10(-2) M. By changing the volumes of the injected sample and the reagent, different acids as well as different concentration ranges of the acids can be titrated without any other adjustments in the SIA manifold or the titration protocol.     

X-ray and scanning electron microscopy archaeometric studies of pigments from the Aguada culture, Argentina

X-ray diffraction (XRD) and scanning electron microscopy (SEM) with energy-dispersive system (EDX) were used in order to obtain mineralogical and chemical composition of white and reddish pigments belonging to the Ambato style of “Aguada” culture, found in the archaeological site of Piedras Blancas (Catamarca, Argentina 500–1100 AD). These pigments are associated with different sectors, two of them being related to funerary context. Due the scarce amount of samples available, it was necessary to develop a new methodology for their study. X-ray diffraction spectra were collected using a low background Si sample holder, which allows the study of small sample amounts (a few milligrams). The mineral quantifications were carried out by applying the Rietveld method to the XRD spectra. The major difficulties arose for reddish pigments, since they contain iron-bearing phases, such as ferruginous clays, in which neither the concentration of Fe^{+ 2} relative to Fe^{+ 3} nor the location in the lattice (occupancy factor) is completely known. With the aim of performing quantitative elemental analysis from SEM-EDX spectra, a special sample holder for the small amounts of available samples was developed. Commercial standards were used in the quantification process and the characteristic intensities were corrected for matrix effects. Micrographs and EDX point spectra allowed the characterization of minor phases and particle analysis. The Rietveld method combined with the new procedure for EDX analysis has proven to be a suitable method for routine quantitative analysis of small amounts of archaeological pigments.     

Capillary electrophoresis with contactless conductivity detection for the quantification of fluoride in lithium ion battery electrolytes and in ionic liquids—A comparison to the results gained with a fluoride ion‐selective electrode

In this study, an optimized method using capillary electrophoresis (CE) with a direct contactless conductivity detector (C⁴D) for a new application field is presented for the quantification of fluoride in common used lithium ion battery (LIB) electrolyte using LiPF₆ in organic carbonate solvents and in ionic liquids (ILs) after contacted to Li metal. The method development for finding the right buffer and the suitable CE conditions for the quantification of fluoride was investigated. The results of the concentration of fluoride in different LIB electrolyte samples were compared to the results from the ion‐selective electrode (ISE). The relative standard deviations (RSDs) and recovery rates for fluoride were obtained with a very high accuracy in both methods. The results of the fluoride concentration in the LIB electrolytes were in very good agreement for both methods. In addition, the limit of detection (LOD) and limit of quantification (LOQ) values were determined for the CE method. The CE method has been applied also for the quantification of fluoride in ILs. In the fresh IL sample, the concentration of fluoride was under the LOD. Another sample of the IL mixed with Li metal has been investigated as well. It was possible to quantify the fluoride concentration in this sample.     

Multiresidue fully automated online SPE-HPLC-MS/MS method for the quantification of endocrine-disrupting and pharmaceutical compounds at trace level in surface water

The present work describes the development and validation of a sensitive method for the determination of traces of diverse groups of pharmaceuticals and endocrine disruptors in surface water. Thirty-seven substances have been selected, including 10 pesticides, 6 hormonal steroids and assimilates, 12 pharmaceuticals, 5 alkylphenols, 1 chlorophenol and 3 other well-known human contaminants, 1 UV filter and 2 plasticisers. An automated online solid-phase extraction (SPE) is directly coupled to liquid chromatography–tandem mass spectrometry. Different SPE columns have been tested, and the injection volume has been optimised. The developed analytical methodology is based on the direct injection of 2.5 mL of water sample acidified at pH 1.6 on an Oasis HLB loading column (20 × 2.1 mm) with 5-µm particles. Then, the chromatographic separation is achieved on a Kinetex XB C18 (100 × 2.1 mm; 1.7 µm) column, and the quantification is realised in multiple-reaction monitoring mode. The online SPE step warrants minimal sample handling, low solvent consumption, high sample throughput, saving time and costs. This method allows the quantification of the target analytes in the lower ng/L concentration range, with limits of quantification (LQs) between 100 pg/L and 10 ng/L, 26 compounds having LQ lower than 1 ng/L. The monitoring of two selected MS/MS transitions for each compound allows the reliable confirmation of positive findings even at the LQ level. The developed and validated methodology has been applied to the analysis of various real samples from two French rivers. Twelve target compounds have been detected in the environmental samples, and the major pollutants are pharmaceuticals usually used by humans (paracetamol, carbamazepine, oxazepam, ketoprofen, trimethoprim). The pesticides atrazine and carbendazim have been ubiquitously detected in real samples too. Metronidazole, sulfamethoxazole and diuron were also frequently quantified in the water samples.