Identification of isobaric amino-sulfonamides without prior separation.

RATIONALE: Direct analysis mass spectrometry (DAMS) techniques offer increased speed of analysis without the need for sample preparation or prior separation. A feature of these techniques is that all ionisable species will typically be analysed at the same time which makes the ability to distinguish between isobaric compounds increasingly important. METHODS: Investigations have been carried out to distinguish isomeric compounds by mass spectrometry only, without the use of any separation technique, in order to further understand the capabilities of DAMS techniques. The work focused on commercially available isomeric amino-sulfonamides, i.e. sulfalene, sulfameter, sulfamethoxypyridazine, sulfamonomethoxine, sulfadoxine, sulfadimethoxine, sulfisomidine, sulfamethazine, sulfamerazine, sulfaperine, sulfadiazine and sulfapyrazine. RESULTS: All the isomeric compounds investigated could be distinguished from each other based on their tandem mass (MS/MS) spectrum or failing that, based on their MS(3) spectrum. Common fragmentation patterns/pathways were observed for groups of the sulfonamides and a rationale for the fragmentations observed is proposed. For the sulfonamides which contain a methoxy group on the pyrimidinyl, pyridazynil, or pyrazinyl ring, the fragmentation-directing feature is the positioning of the methoxy group in the ortho position of the ring with respect to the sulfonamide bond. The presence of an ortho substituent precludes the formation of the product ion resulting from the loss of aniline. CONCLUSIONS: This work has demonstrated the usefulness of MS(n) fragmentation data in identifying and distinguishing isobaric structural isomers without the need for separation by high-performance liquid chromatography (HPLC), allowing the identification of compounds by DAMS techniques. This work has also highlighted patterns in the product ion data which has led to a postulation of how the protonation preference of a molecule can affect the product ions observed and how the presence of ortho substituents can affect this initial protonation preference.

Application of laser ablation multicollector inductively coupled plasma mass spectrometry for the measurement of calcium and lead isotope ratios in packaging for discriminatory purposes.

The potential of high-precision calcium and lead isotope ratio measurements using laser ablation coupled to multicollector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) to aid distinction between four genuine and five counterfeit pharmaceutical packaging samples and further classification of counterfeit packaging samples has been evaluated. We highlight the lack of reference materials for LA-MC-ICP-MS isotope ratio measurements in solids. In this case the problem is minimised by using National Institute of Standards and Technology Standard Reference Material (NIST SRM) 915a calcium carbonate (as solid pellets) and NIST SRM610 glass disc for sample bracketing external standardisation. In addition, a new reference material, NIST SRM915b calcium carbonate, has been characterised in-house for Ca isotope ratios and is used as a reference sample. Significant differences have been found between genuine and counterfeit samples; the method allows detection of counterfeits and aids further classification of packaging samples. Typical expanded uncertainties for measured-corrected Ca isotope ratio values ((43)Ca/(44)Ca and (42)Ca/(44)Ca) were found to be below 0.06% (k = 2, 95% confidence) and below 0.2% for measured-corrected Pb isotope ratios ((207)Pb/(206)Pb and (208)Pb/(206)Pb). This is the first time that Ca isotope ratios have been measured in packaging materials using LA coupled to a multicollector (MC)-ICP-MS instrument. The use of LA-MC-ICP-MS for direct measurement of Ca and Pb isotopic variations in cardboard/ink in packaging has definitive potential to aid counterfeit detection and classification.

Near-infrared hyperspectral unmixing based on a minimum volume criterion for fast and accurate chemometric characterization of counterfeit tablets.

A rapid detection of the nonauthenticity of suspect tablets is a key first step in the fight against pharmaceutical counterfeiting. The chemical characterization of these tablets is the logical next step to evaluate their impact on patient health and help authorities in tracking their source. Hyperspectral unmixing of near-infrared (NIR) image data is an emerging effective technology to infer the number of compounds, their spectral signatures, and the mixing fractions in a given tablet, with a resolution of a few tens of micrometers. In a linear mixing scenario, hyperspectral vectors belong to a simplex whose vertices correspond to the spectra of the compounds present in the sample. SISAL (simplex identification via split augmented Lagrangian), MVSA (minimum volume simplex analysis), and MVES (minimum-volume enclosing simplex) are recent algorithms designed to identify the vertices of the minimum volume simplex containing the spectral vectors and the mixing fractions at each pixel (vector). This work demonstrates the usefulness of these techniques, based on minimum volume criteria, for unmixing NIR hyperspectral data of tablets. The experiments herein reported show that SISAL/MVSA and MVES largely outperform MCR-ALS (multivariate curve resolution-alternating least-squares), which is considered the state-of-the-art in spectral unmixing for analytical chemistry. These experiments are based on synthetic data (studying the effect of noise and the presence/absence of pure pixels) and on a real data set composed of NIR images of counterfeit tablets.

Detection of counterfeit antiviral drug Heptodin and classification of counterfeits using isotope amount ratio measurements by multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) and isotope ratio mass spectrometry (IRMS).

Isotope ratio mass spectrometry (IRMS) and multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) are highly important techniques that can provide forensic evidence that otherwise would not be available. MC-ICP-MS has proved to be a very powerful tool for measuring high precision and accuracy isotope amount ratios. In this work, the potential of combining isotope amount ratio measurements performed by MC-ICP-MS and IRMS for the detection of counterfeit pharmaceutical tablets has been investigated. An extensive study for the antiviral drug Heptodin has been performed for several isotopic ratios combining MC-ICP-MS and an elemental analyser EA-IRMS for stable isotope amount ratio measurements. The study has been carried out for 139 batches of the antiviral drug and analyses have been performed for C, S, N and Mg isotope ratios. Authenticity ranges have been obtained for each isotopic system and combined to generate a unique multi-isotopic pattern only present in the genuine tablets. Counterfeit tablets have then been identified as those tablets with an isotopic fingerprint outside the genuine isotopic range. The combination of those two techniques has therefore great potential for pharmaceutical counterfeit detection. A much greater power of discrimination is obtained when at least three isotopic systems are combined. The data from these studies could be presented as evidence in court and therefore methods need to be validated to support their credibility. It is also crucial to be able to produce uncertainty values associated to the isotope amount ratio measurements so that significant differences can be identified and the genuineness of a sample can be assessed.

Determination of the composition of counterfeit Heptodin tablets by near infrared chemical imaging and classical least squares estimation.

According to the WHO definition for counterfeit medicines, several categories can be established, e.g., medicines containing the correct active pharmaceutical ingredient (API) but different excipients, medicines containing low levels of API, no API or even a substitute API. Obviously, these different scenarios will have different detrimental effects on a patient's health. Establishing the degree of risk to the patient through determination of the composition of counterfeit medicines found in the market place is thus of paramount importance. In this work, classical least squares was used for predicting the composition of counterfeit Heptodin tablets found in a market survey. Near infrared chemical imaging (NIR-CI) was used as a non-destructive measurement technique. No prior knowledge about the origin and composition of the tablets was available. Good API (i.e., lamivudine) predictions were obtained, especially for tablets containing a high API (close to the authentic) dose. Concentration maps of each pure material, i.e., the API (lamivudine) and the excipients microcrystalline cellulose, sodium starch glycollate, rice starch and talc, were estimated. Below 1% of the energy was not explained by the model (residuals percentage) for every pixel in all 12 counterfeit tablets. The similarities among tablets with respect to the total API percentage determined, as well as the corresponding concentration maps, support the classification of the tablets into the different groups obtained in previous work.

Investigation into classification/sourcing of suspect counterfeit Heptodintrade mark tablets by near infrared chemical imaging.

Near infrared chemical imaging (NIR-CI) analysis was performed on 55 counterfeit Heptodin tablets obtained from a market survey and an additional 11 authentic Heptodin tablets for comparison. The aim of the study was to investigate whether NIR-CI can be used to detect the counterfeit tablets and to classify/source them so as to understand the possible number of origins to aid investigators and authorities to shut down counterfeiting operations. NIR-CI combined with multivariate analysis is particularly suited to compare chemical and physical properties of samples, since it is a quick and non-destructive method of analysis. Counterfeit tablets were easily distinguished from the authentic ones. Principal component analysis (PCA) and k-means clustering were performed on the data set. The results from both analyses grouped the counterfeit tablets in 13 main groups. The main groups found with both methods were quite consistent. Out of the 55 tablets only 18% contained the correct active pharmaceutical ingredient (API), i.e., the anti-viral drug lamivudine. The remaining 82% of counterfeit tablets contained talc and starch as main excipients. The API containing tablets classified into three main groups, based mainly on the amount of lamivudine present in the tablet. The group which had close to the correct amount of lamivudine sub-classified into three groups. From the analysis carried out, it is likely that the counterfeit tablets originate from as many as 15 different sources.

Trace level impurity method development with high-field asymmetric waveform ion mobility spectrometry: systematic study of factors affecting the performance.

For the determination of trace level impurities, analytical chemists are confronted with complex mixtures and difficult separations. New technologies such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) have been developed to make their work easier; however, efficient method development and troubleshooting can be quite challenging if little prior knowledge of the factors or their settings is available. We present the results of an investigation performed in order to obtain a better understanding of the FAIMS technology. The influence of eight factors (polarity of dispersion voltage, outer bias voltage, total gas flow rate, composition of the carrier gas (e.g. %He), outer electrode temperature, ratio between the temperatures of the inner and outer electrodes, flow rate and composition of the make-up mobile phase) was assessed. Five types of responses were monitored: value of the compensation voltage (CV), intensity, width and asymmetry of the compensation voltage peak, and resolution between two peaks. Three types of studies were performed using different test mixtures and various ionisation modes to assess whether the same conclusions could be drawn across these conditions for a number of different types of compounds. To extract the maximum information from as few experiments as possible, a Design of Experiment (DoE) approach was used. The results presented in this work provide detailed information on the factors affecting FAIMS separations and therefore should enable the user to troubleshoot more effectively and to develop efficient methods.

Development, validation and transfer into a factory environment of a liquid chromatography tandem mass spectrometry assay for the highly neurotoxic impurity FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine) in paroxetine active pharmaceutical ingredient (API).

This work describes the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for a highly toxic impurity, FMTP (4-(4-fluorophenyl)-1-methyl-1,2,3,6-tetrahydropyridine), in paroxetine active pharmaceutical ingredient (API), followed by the subsequent validation of the methodology and transfer into a global production/quality control environment. The method was developed to achieve a detection limit of 10ppb mass fraction of FMTP in paroxetine API. An LC-MS/MS method was chosen because it provided the required sensitivity and selectivity with minimal sample preparation. This paper discusses the issues with transferring such complex methodology to a production environment. Linearity, repeatability and reproducibility of the method were demonstrated. This work shows that it is possible using the same approach that would be used for the transfer of any analytical method from R&D to a manufacturing environment.

"In-source" fragmentation of an isobaric impurity of lamotrigine for its measurement by liquid chromatography tandem mass spectrometry after pre-concentration using solid phase extraction.

An analytical method has been developed for trace analysis (i.e. sub-ppm levels) of a key synthetic impurity, 14W80 ((Z)-2-(2,3-dichlorophenyl)-2-(guanidinylimino)acetonitrile) in lamotrigine (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine). 14W80 is isobaric with lamotrigine, which gives an extra layer of complexity to its determination and the various problems associated with development of an appropriate methodology are discussed in this work. Ultimately, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method using in-source fragmentation with Atmospheric Pressure Chemical Ionisation (APCI) followed by multiple reaction monitoring (MRM) has been found to provide adequate sensitivity and specificity. A detection limit of 25 ppb mass fraction relative to lamotrigine was achieved for 14W80. The use of solid phase extraction (SPE) enhanced the detection limit to 2 ppb mass fraction relative to lamotrigine.

Investigation of the advanced functionalities of a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer.

Time-of-flight mass spectrometry (ToF-MS) has gained wide acceptance in many fields of chemistry, proteomics, metabolomics and small molecule analysis. ToF-MS, however, has some inherent advantages and drawbacks. Numerous developments have been made to hybrid ToF instruments to improve their capabilities. We have used a quadrupole orthogonal acceleration ToF (Q-oa-ToF) instrument to assess developments made to improve resolution, dynamic range and signal-to-noise (S/N) ratios (i.e. sensitivity). Higher mass resolution can improve the analysis of mixtures containing compounds with similar m/z values and improved mass accuracy gives greater confidence for structural elucidation applications. Wide dynamic ranges are necessary for the analysis of unknown samples or samples that vary widely in analyte concentrations. The performance of the advanced functionalities for routine structural elucidation in terms of resolution, dynamic range and S/N ratios was investigated using test compounds. The results presented in this work demonstrate and validate the use of these new enhancements for Q-ToF instruments and also show their limitations.

Investigation into the factors affecting accuracy of mass measurements on a time-of-flight mass spectrometer using Design of Experiment.

The results of an investigation of the parameters which have the most significant effect on the accuracy of mass measurements on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (q-oaToF) are reported. The influence of eight factors is investigated: ion abundances of reference and analyte compounds, mass difference between analyte and reference compounds, quality of calibration, number of reference acquisitions averaged and TDC (time-to-digital converter) settings (resolution, Np multiplier (number of pushes correction factor), minimum number of points, i.e. minimum acquisition width which defines a peak). To extract the maximum information from as few experiments as possible, a Design of Experiment approach was used. The data will be used as a basis for developing guidance on accurate mass measurement on q-oaToF instruments.

'On-the-fly' hydrogen/deuterium exchange liquid chromatography/mass spectrometry using a dual-sprayer atmospheric pressure ionisation source.

In-source 'on-the-fly' hydrogen/deuterium (H/D) exchange liquid chromatography mass spectrometry (LC/MS) has been investigated. The work was performed using a dual-sprayer source. The analyte was introduced through an electrospray ionisation sprayer and D2O was introduced through an atmospheric pressure chemical ionisation sprayer. To achieve H/D exchange sufficient to determine the number of exchangeable H atoms of a compound, a saturated 'steady-state' D2O atmosphere had to be created in the ion source by having a 2:1 or higher D2O-to-analyte flow rate ratio. Under these conditions H/D exchange levels of 32-90% were achieved. In most cases the H/D exchange was sufficient to measure the number of exchangeable H atoms in some antiulcerative and anthelmintic pharmaceuticals. The concept of in-source 'on-the-fly' H/D exchange by introducing the deuterating agent via a second sprayer has been shown. It allows the integrity of the chromatographic separation to be kept, since the H/D exchange takes place post-separation.

Fragmentation of trimethoprim and other compounds containing alkoxy-phenyl groups in electrospray ionisation tandem mass spectrometry.

This work describes electrospray ionisation tandem mass spectrometry studies of trimethoprim and a series of structurally similar compounds containing alkoxy-phenyl groups; using accurate mass measurement to confirm the proposed fragmentations. Radical cations were observed in the spectra obtained for some of the compounds, as well as uncommon fragmentations showing losses of CH4 and C2H6, whereas other compounds showed the formation of even electron ions. Possible structures for the fragment ions have been proposed and explanations for the different types of fragmentations based on the structures of the compounds. In addition an alternate structure for a fragment ion previously reported for tandem mass spectrometry of trimethoprim has been proposed, based on accurate mass measurement.

Derivatisation of carboxylic acid groups in pharmaceuticals for enhanced detection using liquid chromatography with electrospray ionisation tandem mass spectrometry.

Tris(2,4,6-trimethoxyphenyl)phosphonium propylamine bromide (TMPP) has been used for the derivatisation of maleic, fumaric, sorbic and salicylic acids to facilitate determination using liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) in positive ion mode. Detection limits, achieved using multiple reaction monitoring mode, were 2, 4, 0.4 and 540 fmol (5 muL injection) for derivatised fumaric, sorbic, maleic and salicylic acids, respectively. In comparison, detection limits achieved in negative ion mode for the underivatised acids were 24, 51, 2, and 117 fmol, respectively. The method was successfully used for the determination of sorbic acid in a sample of Panadol. The derivatisation of salicylic acid was not as successful, probably due to poor reaction efficiency.

Component detection weighted index of analogy: similarity recognition on liquid chromatographic mass spectral data for the characterization of route/process specific impurities in pharmaceutical tablets.

Detection and identification of impurities in pharmaceuticals is an essential task for determining the possible infringement of a patent. This article reports a multivariate analysis method to distinguish between tablets of the same substance on the basis of their origin, by characterizing route/process specific impurities via diagnostic ion chromatograms, using liquid chromatography/mass spectrometry (LC/MS). The approach is based on the formulation of a novel index that quantifies the similarity between LC/MS samples, named the component detection weighted index of analogy. The index estimates similarity by fully exploiting the two-dimensional nature of the data, where the relative contribution of chromatograms relates to their quality and noise level. Results show that well-defined clusters are formed according to the origin of tablets; a series of ions are identified as characterizing each class and can be used to predict the origin of unknown tablet samples. The method presented is designed for analysis of larger data sets and can be suitable for exploratory analysis where any a priori knowledge on the data is scarce or absent, hence requiring the acquisition of chromatograms in a broad m/z range.

Analysis of betamethasone, dexamethasone and related compounds by liquid chromatography/electrospray mass spectrometry.

A reversed-phase high-performance liquid chromatography/electrospray ionisation mass spectrometry (HPLC/ESI-MS) method has been developed to conclusively differentiate the epimers betamethasone and dexamethasone and various esterification products (betamethasone and dexamethasone 21-acetate, betamethasone and dexamethasone 21-phosphate, betamethasone 17-valerate, betamethasone 21-valerate and betamethasone 17,21-dipropionate) in counterfeit drugs. Good separation with baseline resolution of all epimers or isomers was obtained on a Zorbax Eclipse XDB or Luna C8 column, using a step gradient with mobile phases of 0.05 M ammonium acetate and acetonitrile. Betamethasones can also be distinguished by the relative abundance of their m/z 279 ion in the positive electrospray tandem mass spectra. The LC/MS or LC/MS/MS method developed was successfully applied to the analysis of drug product samples, i.e. creams and tablets.

Identification of the 'wrong' active pharmaceutical ingredient in a counterfeit Halfan drug product using accurate mass electrospray ionisation mass spectrometry, accurate mass tandem mass spectrometry and liquid chromatography/mass spectrometry.

Methodology is presented for identifying an unknown active (pharmaceutical) ingredient (AI) in a counterfeit drug product. A range of mass spectrometric techniques, i.e., accurate mass mass spectrometry, tandem mass spectrometry (MS/MS) and liquid chromatography/mass spectrometry (LC/MS), has been employed to determine the AI in a counterfeit Halfan suspension, an antimalarial drug. In particular, use of LockSpray accurate mass MS/MS allowed identification of parts of the molecule from fragments, hence limiting the number of possible elemental compositions for the nominal mass of 278 found for the AI in the counterfeit product. The analysis of the isotope pattern observed for the protonated molecule further reduced the number of possible elemental compositions. A literature search for readily commercially available compounds of molecular formula C(12)H(14)N(4)O(2)S suggested that the AI was either sulfamethazine or sulfisomidine. An LC/MS separation of those two compounds and reference MS/MS spectra obtained for sulfamethazine and sulfisomidine led to the conclusion that the AI in the counterfeit Halfan suspension is sulfamethazine, which is an antibacterial agent.