Simultaneous determination of thirty non-steroidal anti-inflammatory drug residues in swine muscle by ultra-high-performance liquid chromatography with tandem mass spectrometry

An ultra-high-performance liquid chromatography with tandem mass spectrometric detection (UHPLC–MS/MS) method was established for the simultaneous determination of residues of thirty non-steroidal anti-inflammatory drugs (NSAIDs) in swine muscle. The samples were extracted with acetonitrile and phosphoric acid. The extracts were defatted with n-hexane, and then purified by HLB solid-phase extraction cartridge. Analysis was carried out on UHPLC–ESI-MS/MS working with multiple reaction monitoring mode with polarity switching. Limits of detection were between 0.4 μg/kg and 2.0 μg/kg, and limits of quantification were between 1.0 μg/kg and 5.0 μg/kg. The recoveries of NSAIDs were between 61.7% and 125.7% at spiked levels of 1.0–500 μg/kg. The repeatability was less than 8% and the within-laboratory reproducibility was not more than 12.3%. The method was reliable, convenient and sensitive.     

Ultra-performance liquid chromatography coupled to linear ion trap mass spectrometry for the identification of drug metabolites in biological samples

The coupling of ultra-performance liquid chromatography, operating at elevated pressures, to a linear ion trap mass spectrometer provides a high-performance system suitable for drug metabolite characterisation. This system demonstrates improved chromatographic efficiency and sensitivity and at the same time provides diagnostic MSn data often critical for metabolite structural assignment. The linear ion trap was capable of dealing with the high chromatographic efficiencies and hence narrow peak widths associated with 1.7 microm particle-packed column separations. Polarity switching and data-dependent MSn data were generated with ease, and applied to the identification of metabolites found in human plasma.     

Confirmatory analysis of non-steroidal anti-inflammatory drugs in bovine milk by high-performance liquid chromatography with fluorescence detection

HPLC with fluorescence detection is considered for confirmatory analysis of group B veterinary drugs by the European Union legislation. A procedure for confirming the presence of anti-inflammatory non-steroidal drug (NSAID) residues in bovine milk by reversed phase high-performance liquid chromatography with fluorescence detection is herein described. The native fluorescence of nine drugs belonging to different NSAID sub-classes, namely flurbiprofen, carprofen, naproxen, vedaprofen, 5-hydroxy-flunixin, niflumic acid, mefenamic acid, meclofenamic acid and tolfenamic acid, allowed for detection in bovine milk down to 0.25–20.0 μg/kg. Confirmation of the nine NSAIDs is attained by fluorescence detection at characteristic excitation and emission wavelengths. The procedure described is simple and selective. Limits of quantification (LOQs) ranging between 0.25 and 20 μg/kg were measured; satisfactory trueness and within-laboratory reproducibility data were calculated at LOQ spiking levels, apart from 5-hydroxy-flunixin. The procedure developed is used in our laboratory for confirmation of each one of the above mentioned NSAIDs in bovine milk, to support results after HPLC quantitative analysis with UV–vis detection.     

Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromatography coupled to Orbitrap and liquid chromatography coupled to tandem mass spectrometry

A simple method for the determination of some anthelmintic drugs and phenylbutazone residues in milk and muscle was developed. Following a fast and easy extraction and evaporation procedure, the extract was injected into an ultra performance liquid chromatography system coupled to a single stage Orbitrap detector. The high mass resolution of 50,000 full width at half maximum and corresponding narrow mass windows permitted a very selective and sensitive detection of analytes without requiring fragmentation of the observed [M+H]⁺ or [M+Na]⁺ ions. This eliminated some difficulties which have plagued the analysis of compounds belonging to the group of avermectins. The analytical method was validated according to the EU commission decision for Orbitrap based, but also for more traditional tandem mass spectrometry based detection and quantification. Equal repeatability but significantly higher sensitivity for critical compounds (avermectins) was obtained for the Orbitrap based detection. A result of this study was the conclusion that analytes with poor fragmentation properties (e.g. sodium-cationized molecules) can be more easily quantified by single stage high resolution mass spectrometry than by tandem mass spectrometry.     

Investigation of amodiaquine bulk drug impurities by liquid chromatography/ion trap mass spectrometry

Three unknown impurities in an amodiaquine bulk drug sample were detected by reversed-phase high-performance liquid chromatography with ultraviolet detection (HPLC/UV). A liquid chromatography/tandem mass spectrometry (LC/MS(n)) method is described for the investigation of these impurities. Mass spectral data were acquired on an LCQ ion trap mass analyzer equipped with an electrospray ionization (ESI) source operated in positive ion mode. The fragmentation behavior of amodiaquine and its impurities has been studied. Based on the mass spectral data and the specifics of the synthetic route, the possible structures of these impurities were elucidated as 4-[(5-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)phenol (impurity I), 4-[(7-chloroquinolin-4-yl)-amino]phenol (impurity II) and 4-[(7-chloroquinolin-4-yl)amino]-2-(diethylaminomethyl)-N(1)-oxy]phenol (impurity III). The structures were confirmed by their independent synthesis and NMR spectral assignment.     

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.     

Direct detection of yessotoxin and its analogues by liquid chromatography coupled with electrospray ion trap mass spectrometry

A liquid chromatography mass spectrometry (LC-MS) method is proposed for the sensitive, specific and direct detection of yessotoxin and its analogues, marine biotoxins which are associated with diarrhetic shellfish poisoning (DSP) and which have been found in the North Adriatic sea since 1995. The LC-MS method provided a detection limit of 70 pg for yessotoxin in full scan mode and was applied to determine the toxic profiles of a number of extracts or partially purified fractions of toxic mussels collected along the Emilia Romagna coasts (Italy) in the period 1995-1999. Detection of a desulfo-yessotoxin derivative from Mytilus galloprovincialis collected in 1998 is also reported.     

Rapid confirmatory analysis of non-steroidal anti-inflammatory drugs in bovine milk by rapid resolution liquid chromatography tandem mass spectrometry

A rapid method has been developed to analyse carprofen (CPF), diclofenac (DCF), mefenamic acid (MFN), niflumic acid (NIFLU), naproxen (NAP), oxyphenylbutazone (OXYPHEN), phenylbutazone (PBZ) and suxibuzone (SUXI) residues in bovine milk. Milk samples are extracted with acetonitrile and sample extracts were purified on Evolute™ ABN solid phase extraction cartridges. Aliquots were analysed by rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) with a runtime of 6.5 min. The method was validated in bovine milk, according to the criteria defined in Commission Decision 2002/657/EC. CCα values of 0.46, 1.08, 0.92, 1.26, 1.29, 2.12, 0.55 and 2.86 ng mL⁻¹ were determined for CPF, DCF, MFN, NIFLU, NAP, OXYPHEN, PBZ and SUXI, respectively. CCβ values of 0.79, 1.85, 1.56, 2.15, 2.19, 3.62, 0.94 and 4.87 ng mL⁻¹ were determined for CPF, DCF, MFN, NIFLU, NAP, OXYPHEN, PBZ and SUXI, respectively. The measurement uncertainty of the method was estimated at 9, 28, 28, 45, 46, 45, 10 and 39% for CPF, DCF, MFN, NIFLU, NAP, OXYPHEN, PBZ and SUXI. Fortifying bovine milk samples (n = 18) in three separate assays, show the accuracy of the method to be between 82 and 108%. The precision of the method, expressed as RSD values for the within-lab reproducibility at the three levels of fortification (5, 7.5 and 10 ng mL⁻¹) was less than 16%, respectively. The advantage of the method is that low ng mL⁻¹ levels can be detected and quantitatively confirmed rapidly in milk and that 3 batches of samples can be analysed within a single day using RRLC-MS/MS with a runtime of 6.5 min.     

Analysis of unknown compounds in gentamicin bulk samples with liquid chromatography coupled with ion trap mass spectrometry

Six unknown compounds present in bulk gentamicin samples have been identified by ion-pairing reversed-phase liquid chromatography coupled with ion trap mass spectrometry. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of the available related substances and gentamicin reference substances. Seven other unknown components were partially identified.     

Characterization of dihydrostreptomycin‐related substances by liquid chromatography coupled to ion trap mass spectrometry

Dihydrostreptomycin sulphate (DHS) is a water‐soluble, broad‐spectrum aminoglycoside antibiotic. For quantitative analysis, the European Pharmacopoeia (Ph. Eur.) prescribes an ion‐pairing liquid chromatography/ultraviolet (LC/UV) method using a C18 stationary phase. Several unknown compounds were detected in commercial samples. Hence, for characterization of these unknown peaks in a commercial DHS sample, the Ph. Eur. method was coupled to mass spectrometry (MS). However, since the Ph. Eur. method uses a non‐volatile mobile phase, each peak eluted was collected and desalted before introduction into the mass spectrometer. The desalting procedure was applied to remove the non volatile salt, buffer and ion‐pairing reagent in the collected fraction. In total, 20 impurities were studied and 14 of them were newly characterized. Five impurities which are already reported in the literature were also traced in this LC/UV method. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of levamisole in feeds by liquid chromatography coupled to electrospray mass spectrometry on an ion trap

Test methods have to be developed by laboratories for official control to monitor possible misuse of veterinary drugs in animal productions, also through feeding stuff. A novel method for identification and quantification of levamisole in feeds by liquid chromatography coupled to electrospray mass spectrometry in an ion trap (LC/ESI‐MS/MS) is herein described; after a single‐step cleanup by liquid‐liquid extraction from the feed and separation by reversed‐phase liquid chromatography, levamisole was determined and unambiguously confirmed by tandem mass spectrometry, on the basis of two product ions. The method was in‐house validated, according to the Regulation 882/2004/EC, evaluating trueness, repeatability, within‐laboratory reproducibility, ruggedness, specificity, and the limit of quantification (LOQ). The method is reliable and specific for complete and complementary feeds for pigs, cattle, rabbits and poultry; very good mean recoveries (higher than 92 %) and precision (RSD values < 15.2%) were attained. The LOQ at 2.0 mg/kg was verified. Moreover, we describe how the method was developed to support Italian Police investigations regarding illegal treatments of pigs; in this case, since the drug(s) added to the feed were unknown, a preliminary untargeted analysis was performed by full scan mass spectrometry on an ion trap, from 50 up to 2000 m/z; the presence of levamisole was hypothesised, on the basis of the most abundant ion and its fragmentation pattern. Then, levamisole was unambiguously confirmed by the ion trap LC/ESI‐MS/MS method. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of unknown compounds in azithromycin bulk samples with liquid chromatography coupled to ion trap mass spectrometry

A selective reversed-phase liquid chromatography/mass spectrometry (LC/MS(n)) method is described for the identification of azithromycin impurities and related substances in commercial azithromycin samples. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an atmospheric pressure chemical ionization interface operated in positive ion mode. The LCQ provides on-line LC/MS(n) capability, making it ideally suited for identification purposes. In comparison with UV detection, this hyphenated technique provides as its main advantage efficient identification of novel substances without time-consuming isolation and purification procedures. Using this technique, six novel related substances detected in commercial azithromycin samples have been studied.     

Characterization of impurities in dirithromycin by liquid chromatography/ion trap mass spectrometry

With a recently developed liquid chromatographic (LC) method, using a phosphate buffer, several unknown impurities present in dirithromycin samples were separated. In this paper, a reversed-phase liquid chromatography-tandem mass spectrometry method is described for the investigation of dirithromycin and related substances. The method employed uses a Zorbax Extend C18 column (250 mm x 4.6 mm I.D.), 5 microm, and a mobile phase consisting of acetonitrile, 2-propanol, water and ammonium acetate solution pH 8.5. Mass spectral data are acquired on an LCQ ion trap mass spectrometer equipped with an electrospray ion (ESI) source operated in the positive ion mode. The LCQ is ideally suited for the identification of related substances because it provides on-line LC/MS(n) capability, which allows efficient identification without time-consuming isolation and purification procedures. Using this method, the fragmentation behavior of dirithromycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total the structures of nine impurities were elucidated, among which three were different analogues with a modification in the side chain on the oxazine ring. Two impurities showed a different alkyl group in position C13. In two impurities the desosamine sugar was involved with changes in the degrees of methylation of the amino group. One unknown impurity was identified as dirithromycin F and another unknown was characterized as dirithromycin N-oxide.     

Determination of cylindrospermopsin in freshwaters and fish tissue by liquid chromatography coupled to electrospray ion trap mass spectrometry

Cylindrospermopsin (CYN) is a toxic alkaloid‐like compound produced by some strains of cyanobacteria, procariotic organisms occurring in water blooms, observed worldwide in eutrophic lakes and drinking water reservoirs. Methods for determination of CYN in freshwater and fish muscle by liquid chromatography coupled to electrospray ion trap mass spectrometry are herein described. The performances of both methods are reported; ion trap LC/ESI‐MS/MS resulted highly selective and reliable in unambiguous identification of CYN, based on monitoring the precursor ion and three product ions. The methods developed showed satisfactory mean recoveries (higher than 63.6%) and relative standard deviations, ranging from 5.8 to 9.8%. The limits of quantification at 0.10 ng/mL in freshwaters and 1.0 ng/g in fish muscle, respectively, allow for determination of CYN also in early contamination stages. Ion trap LC/ESI‐MS/MS was successfully applied to the identification and quantification of CYN in water and cyanobacteria extracts from Lake Averno, near Naples, representing the first case of contamination described in southern Italy. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of impurities in spiramycin by liquid chromatography/ion trap mass spectrometry

A reversed-phase liquid chromatography/tandem mass spectrometry method is described for the investigation of spiramycin and related substances. The method uses an XTerra C18 column (250 x 4.6 mm i.d.), 5 microm, and a mobile phase consisting of acetonitrile, methanol, water and ammonium acetate solution, pH 6.5. Mass spectral data were acquired on an LCQ ion trap mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) operated in the positive ion mode. Using this method, the fragmentation behavior of spiramycin and its related substances was studied and the unknown impurities occurring in commercial samples were investigated. In total 17 compounds were identified, among which three reported as specified impurities in the European Pharmacopoeia. The other impurities showed mainly a modification in the forosamine sugar or in the substituent at C-3 and C-6 positions. In one impurity, the mycarose sugar is absent.     

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.     

Multi-residue liquid chromatography/tandem mass spectrometry method for the detection of non-steroidal anti-inflammatory drugs in bovine muscle: optimisation of ion trap parameters

A multi-residue liquid chromatography/tandem mass spectrometry method (LC/MS2) was developed for the detection of the non-steroidal anti-inflammatory drugs acetylsalicylic acid (via the marker residue salicylic acid), flunixin, phenylbutazone, tolfenamic acid, meloxicam and ketoprofen, in bovine muscle. After extraction of the bovine muscle with acetonitrile, the cleanup was performed using a Oasis HLB column. The evaporated eluate was reconstituted and analysed by LC/MS2. To obtain optimal detection of salicylic acid and phenylbutazone, the ion trap mass spectrometric parameters activation q and maximum ion injection time, respectively, were optimised. The activation q for salicylic acid was increased to obtain reliable detection of both salicylic acid and its product ion. The maximum ion injection time for the time segment containing phenylbutazone was decreased since there were not enough scans across the chromatographic peak of this compound. The multi-residue method was able to detect the different analytes below or at the maximum residue limit (MRL) or minimum required performance limit (MRPL) or, in the case of phenylbutazone and ketoprofen, at 100 and 20 microg kg(-1), respectively.     

Screening and identification of multi-component in Qingkailing injection using combination of liquid chromatography/time-of-flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

An approach for screening and identification of multi-component in complex traditional Chinese medicine systems with a combinative LC/MS (MSⁿ) technique was described in this paper. The chemical profile of Qingkailing injection, a well-known traditional Chinese formula in China, was studied using the established method as for an application. Benefit from combining the accurate mass measurement of LC/TOF-MS to generate elemental compositions and the complementary multilevel structural information provided by LC/ion trap MSⁿ, 33 components in Qingkailing injection were identified in all. The three isomers of chlorogenic acid, isochlorogenic acid and neochlorogenic acid which are derived from Flos Lonicerae, one of the medicinal materials in Qingkailing, were differentiated by verifying their MS³ fragmentation data. All the components identified were surveyed and classified according to their medicinal materials derivation. This study is expected to provide an effective and reliable pattern for comprehensive and systematic characterization of the complex traditional Chinese medicine systems.