Gillian McMahon

A review of analytical methods for the determination of aminoglycoside and macrolide residues in food matrices

The development of antibiotic resistance in bacteria has been attributed to the overuse of antimicrobials in human medicine. Another route by which humans are exposed to antibiotics is through the animal foods we eat. In modern agricultural practice, veterinary drugs are being used on a large scale, administered for treating infection or prophylactically to prevent infection. Hence, there is pressure on analytical scientists to detect and confirm the presence of antimicrobials in foods of animal origin. The aminoglycosides and macrolides are two families of antibiotics, each with important applications in veterinary medicine. These antibiotics are widely used in the treatment of bacterial disease, e.g., aminoglycosides for mastitis and macrolides for enteric infections. They have also been used as feed additives for growth promotion. As a result, legislation has been laid down by the European commission in which member states must meet strict criteria for monitoring residues (including antimicrobials). Testing for low levels of aminoglycosides and macrolides in foods is a priority and hence the development of fast, reliable, sensitive methods for their extraction and subsequent analysis is of great interest. This paper reviews analytical methods for both extracting and determining these classes of antibiotics in various food matrices focusing in particular on the last 10 years. Extraction and clean-up methods such as deproteinisation, and solid-phase extraction are described. Various screening methods are also covered including thin layer chromatography (TLC), enzyme immunoassay, capillary electrophoresis (CE) and microbiological assays. Finally, liquid chromatography (LC) methods are discussed which are combined with mass spectrometry (MS) when sensitivity requirements are stringent.

An LC–MS method for the determination of pharmaceutical compounds in wastewater treatment plant influent and effluent samples

Pharmaceuticals are continually introduced into the environment as a result of industrial and domestic use. In recent years they have emerged as environmental pollutants. An analytical method has been developed allowing for simultaneous detection and identification of 20 pharmaceutical compounds from various therapeutic classes using solid phase extraction (SPE) followed by liquid chromatography-electrospray ionisation mass spectrometry (LC-MS/MS). The limits of detection and limits of quantitation for the method were in the ng/L-microg/L range. The method was applied to influent and effluent samples from three wastewater treatment plants (WWTPs). Fifteen compounds were identified in the sample matrix with salicylic acid and ibuprofen being the most abundant at 9.17 and 3.20 microg/L respectively.

Detection of calcium phosphate crystals in the joint fluid of patients with osteoarthritis – analytical approaches and challenges

Clinically, osteoarthritis (OA) is characterised by joint pain, stiffness after immobility, limitation of movement and, in many cases, the presence of basic calcium phosphate (BCP) crystals in the joint fluid. The detection of BCP crystals in the synovial fluid of patients with OA is fraught with challenges due to the submicroscopic size of BCP, the complex nature of the matrix in which they are found and the fact that other crystals can co-exist with them in cases of mixed pathology. Routine analysis of joint crystals still relies almost exclusively on the use of optical microscopy, which has limited applicability for BCP crystal identification due to limited resolution and the inherent subjectivity of the technique. The purpose of this Critical Review is to present an overview of some of the main analytical tools employed in the detection of BCP to date and the potential of emerging technologies such as atomic force microscopy (AFM) and Raman microspectroscopy for this purpose.

Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation.

Background:Mammographic microcalcifications represent one of the most reliable features of nonpalpable breast cancer yet remain largely unexplored and poorly understood.Methods:We report a novel model to investigate the in vitro mineralisation potential of a panel of mammary cell lines. Primary mammary tumours were produced by implanting tumourigenic cells into the mammary fat pads of female BALB/c mice.Results:Hydroxyapatite (HA) was deposited only by the tumourigenic cell lines, indicating mineralisation potential may be associated with cell phenotype in this in vitro model. We propose a mechanism for mammary mineralisation, which suggests that the balance between enhancers and inhibitors of physiological mineralisation are disrupted. Inhibition of alkaline phosphatase and phosphate transport prevented mineralisation, demonstrating that mineralisation is an active cell-mediated process. Hydroxyapatite was found to enhance in vitro tumour cell migration, while calcium oxalate had no effect, highlighting potential consequences of calcium deposition. In addition, HA was also deposited in primary mammary tumours produced by implanting the tumourigenic cells into the mammary fat pads of female BALB/c mice.Conclusion:This work indicates that formation of mammary HA is a cell-specific regulated process, which creates an osteomimetic niche potentially enhancing breast tumour progression. Our findings point to the cells mineralisation potential and the microenvironment regulating it, as a significant feature of breast tumour development.

Development of a high-performance liquid chromatographic-mass spectrometric method for the determination of cellular levels of the tyrosine kinase inhibitors lapatinib and dasatinib.

A highly sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed to quantify cellular levels of the tyrosine kinase inhibitors (TKIs) dasatinib (Sprycel) and lapatinib (Tykerb, Tyverb). Cellular samples were extracted with a tert-butyl methyl ether:acetonitrile (3:1, v/v):1 M ammonium formate pH 3.5 (8:1, v/v) mixture. Separation was achieved on a Hyperclone BDS C18 (150 mm x 2.0 mm 3 microm) column with isocratic elution using a mobile phase of acetonitirile-10 mM ammonium formate, pH 4 (54:46, v/v), at a flow rate of 0.2 mL/min. The TKIs were quantified using a triple quadrupole mass spectrometer which was operated in multi-reaction-monitoring mode employing positive electrospray ionisation. The limit of detection and limit of quantification for lapatinib was determined to be 15 and 31 pg on column, respectively. The limit of detection and quantification for dasatinib was 3 and 15 pg on column, respectively. The method allowed for sensitive and accurate determination of cellular levels of dasatinib and lapatinib. In addition, we examined the potential for this method to be utilised to quantitate other TKIs, using gefitinib, erlotinib, imatinib and sorafenib as examples. In principle, these agents were also quantifiable by this method, however, no drug specific validation studies were undertaken with these TKIs. The data indicates that in the cancer cell-line model, DLKP, significantly more lapatinib accumulates in cells in comparison to dasatinib. Additionally, over-expression of the membrane protein drug transporter, P-glycoprotein (P-gp) a common cancer drug resistance mechanism, greatly reduces the cellular accumulation of dasatinib but not of lapatinib.

Rapid and sensitive liquid chromatography-tandem mass spectrometry for the quantitation of epirubicin and identification of metabolites in biological samples.

A highly sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of epirubicin in serum and cell specimens using daunorubicin as an internal standard. Using atmospheric pressure chemical ionisation (APCI), the epirubicin metabolites were readily distinguishable by their fragmentation pattern in the mass spectrometer. Selected reaction monitoring (SRM) mode was employed for quantitation of epirubicin and the metabolites. Following extraction, chromatography was performed on a C18 column with a mobile phase consisting of water-acetonitrile-formic acid, pH 3.2, with a flow rate of 200mul/min. The limit of detection (LOD) and the limit of quantitation (LOQ) of this method in serum were determined to be 1.0 and 2.5ng/ml, respectively. Linearity of the method was verified over the concentration range of 2.5-2000ng/ml, with a high correlation coefficient (R(2)>/=0.998). For the extraction procedure, an aliquot of 500mul serum, spiked with internal standard, was extracted using a chloroform-2-isopropanol (2:1, v/v) mixture. The method has been applied to the analysis of epirubicin in cancer cell samples and the identification of known and unknown metabolites in clinical trial patient serum samples.

Detection of basic calcium phosphate crystals in osteoarthritis.

Osteoarthritis (OA) is the most common human joint disorder. Its complex pathogenesis remains poorly understood but appears multifactorial. To date, no specific pharmacological agent has been identified to alter the disease course of OA. Calcification of articular cartilage is a hallmark of OA and evidence suggests it contributes directly to joint degeneration. Calcium crystals are frequently found in OA synovial fluid but their exact role in the disease process is unclear. Basic calcium phosphate (BCP) crystals are the predominant crystal type found in OA and recent data indicate a pathogenic role for these crystals in OA. However, information on the exact frequency and distribution of BCP crystals vary considerably, mainly due to the lack of simple and reliable methods of detection. The purpose of this review is to describe the current available methods for detecting BCP crystals and to highlight their obvious advantages and limitations. Recent developments in the field are also discussed with particular reference to their potential clinical applicability. Improved methods of detection for BCP crystals could potentially aid the diagnosis of OA and the development of novel therapies.

Simultaneous determination of efavirenz, rifampicin and its metabolite desacetyl rifampicin levels in human plasma.

A simple and rapid isocratic, high performance liquid chromatography (HPLC) assay employing solid phase extraction (SPE) for the simultaneous determination of the anti HIV drug, efavirenz, the anti-tuberculosis drug, rifampicin and the desacetyl metabolite of rifampicin in plasma from HIV/tuberculosis infected patients has been developed. Using a Zorbax SB-Phenyl reverse-phase analytical column with UV detection, good separation and detection of the drugs was attained within a 10min run time. Intra- and inter-assay precision RSD values were found to be less than 15% at the concentrations examined (0.1-20μg/mL). The LOQ was found to be 0.1μg/mL for each agent and the assay was found to generate a linear response up to 20μg/mL. This low cost assay can accurately detect efavirenz and rifampicin concentrations within a clinically relevant concentration range using standard chromatography equipment, making it particularly applicable to resource-limited settings.

Simultaneous determination of anthracyclines and taxanes in human serum using online sample extraction coupled to high performance liquid chromatography with UV detection

An online SPE-LC method that can determine both anthracyclines and taxanes simultaneously in human serum samples is reported. The entire method of extraction, separation and UV detection was achieved online by column switching between an SPE column (Biotrap 500 (20 x 4 mm)) and an analytical column (Zorbax XDB C18, 150 x 4.6 mm, 5 microm) with a 23 min total cycle time. The method is linear (r(2)>0.998) over the range of 0.5-25 microg/mL. The analytes of interest are retained on the SPE column with good recovery (84-117%), while proteins and other serum components elute to waste. This online clean-up is much faster (150 s) and less manual than traditional off-line extraction methods. Using 0.1 mL spiked serum samples, the LOQ was 0.5 microg/mL. Intra- and inter-day precision were acceptable (< or = 15% RSD) at and above the LOQ. The method was applied to the analysis of serum samples from patients undergoing chemotherapy with these agents.

Determination of calcium in synovial fluid samples as an aid to diagnosing osteoarthritis

BACKGROUND Microscopic inorganic crystals are commonly observed in the synovial fluid of patients suffering from arthritic diseases. Basic calcium phosphate (BCP) crystals are known to occur quite commonly in the joint fluid of osteoarthritis (OA) patients and are insoluble at physiological pH. Current analysis of patient synovial fluid depends on light microscopy and staining with Alizarin Red-S. Both methods cannot identify crystals < 1µm in size and are highly subjective. This article investigates the use of o-cresolphthalein complexone (OCP), a colorimetric reagent, to quantify calcium from crystals isolated from synovial fluid samples as a means of identifying the presence of BCP and, hence, improving the diagnosis of OA. RESULTS Inorganic crystals were isolated following degradation of the biological sample matrix with hyaluronidase. 1-M HNO(3) was used for crystal dissociation into ions and the colorimetric response of OCP to calcium was measured in a basic environment of 2-amino-2-methyl-1-propanol. The average calcium content in OA patient samples was up to 40% higher than in rheumatoid arthritis (RA) patient samples. RA samples were used as a comparison, because they are generally accepted to be crystal free. Within the OA group, higher levels of calcium were detected in three out of 12 synovial fluid samples, which correlated with a significantly greater number of BCP crystals detected during microscopic examination. CONCLUSIONS A simple method based on colorimetry for measurement of calcium content and semiquantification of BCP crystals in synovial fluid samples has been described. Sample pretreatment following addition of hyaluronidase proved to be effective in reducing viscosity and aiding the dissociation of BCP crystals in synovial fluid samples.