Simultaneous analysis of carcinogenic N-nitrosamine impurities in metformin tablets using on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

Contamination of active pharmaceutical ingredients (APIs) and pharmaceutical preparations with carcinogenic N-nitrosamines has led to recalls of these products and supply shortages to patients. The present study describes the development of a highly sensitive method for simultaneous analysis of seven N-nitrosamines using on-line in-tube solid-phase microextraction (IT-SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine their actual contamination in metformin tablets. Using a Carboxen 1006 PLOT capillary as the extraction device for IT-SPME, these compounds were efficiently extracted and concentrated 6‒24-fold by subjecting 40 µL of sample to 25 repeated draw/eject cycles at a rate of 0.2 mL/min. The seven N-nitrosamines were separated within 11 min by gradient elution with 0.1 % formic acid solution and acetonitrile as the mobile phase using a CAPCELL PAK C18 MGII column and detected by multiple reaction monitoring in positive ion mode. The calibration curve showed linearity in the range 0.2‒50 ng/mL and detection limits (S/N = 3) in the range 3‒112 pg/mL. The intra-day and inter-day precisions were less than 5.5 % and 7.0 % (n = 6), respectively, with accuracies ranging from 93‒117 %. Following ultrasonic extraction with water, centrifugation and filtration of the supernatant liquid through a membrane filter, the N-nitrosamine impurities in metformin tablets could be analyzed by IT-SPME/LC‒MS/MS. Their limits of quantification (S/N = 10) were 0.1‒5.1 pg/mg API and recoveries ranged from 87‒102 %. Analysis of eight metformin tablets from eight manufacturers showed that 5.8‒7.5 pg/mg N-nitrosodimethylamine were present in three tablets, with no other N-nitrosamines detected in any of the eight tablets. This method may be useful in testing for N-nitrosamine impurities in pharmaceutical preparations.

Online In-Tube Solid-Phase Microextraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Tobacco-Specific Nitrosamines in Hair Samples.

Active and passive smoking are serious public health concerns Assessment of tobacco smoke exposure using effective biomarkers is needed. In this study, we developed a simultaneous determination method of five tobacco-specific nitrosamines (TSNAs) in hair by online in-tube solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). TSNAs were extracted and concentrated on Supel-Q PLOT capillary by in-tube SPME and separated and detected within 5 min by LC-MS/MS using Capcell Pak C18 MGIII column and positive ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. The calibration curves of TSNAs showed good linearity in the range of 0.5-1000 pg mL-1 using their stable isotope-labeled internal standards. Moreover, the limits of detection (S/N = 3) of TSNAs were in the range of 0.02-1.14 pg mL-1, and intra-day and inter-day precisions were below 7.3% and 9.2% (n = 5), respectively. The developed method is highly sensitive and specific and can easily measure TSNA levels using 5 mg hair samples. This method was used to assess long-term exposure levels to tobacco smoke in smokers and non-smokers.

The L-type amino acid transporter LAT1 inhibits osteoclastogenesis and maintains bone homeostasis through the mTORC1 pathway.

L-type amino acid transporter 1 (LAT1), which is encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types, contributing to the pathogenesis of cancer and neurological disorders. Amino acid substrates of LAT1 have a beneficial effect on bone health directly and indirectly, suggesting a potential role for LAT1 in bone homeostasis. Here, we identified LAT1 in osteoclasts as important for bone homeostasis. Slc7a5 expression was substantially reduced in osteoclasts in a mouse model of ovariectomy-induced osteoporosis. The osteoclast-specific deletion of Slc7a5 in mice led to osteoclast activation and bone loss in vivo, and Slc7a5 deficiency increased osteoclastogenesis in vitro. Loss of Slc7a5 impaired activation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway in osteoclasts, whereas genetic activation of mTORC1 corrected the enhanced osteoclastogenesis and bone loss in Slc7a5-deficient mice. Last, Slc7a5 deficiency increased the expression of nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1) and the nuclear accumulation of NFATc1, a master regulator of osteoclast function, possibly through the canonical nuclear factor κB pathway and the Akt-glycogen synthase kinase 3ß signaling axis, respectively. These findings suggest that the LAT1-mTORC1 axis plays a pivotal role in bone resorption and bone homeostasis by modulating NFATc1 in osteoclasts, thereby providing a molecular connection between amino acid intake and skeletal integrity.

A sensitive method for the determination of tobacco-specific nitrosamines in mainstream and sidestream smokes of combustion cigarettes and heated tobacco products by online in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry.

A simple and sensitive method, using automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed for the determination of four tobacco-specific nitrosamines (TSNAs) in mainstream and sidestream smoke of combustion cigarettes and heated tobacco products. These TSNAs were separated within 4 min on a Capcell Pak C18 MGⅢ column and detected in the positive ion mode by multiple reaction monitoring. The optimum in-tube SPME conditions were 30 draw/eject cycles of 40 µL of sample at a flow rate of 200 µL min-1 using a Supel-Q PLOT capillary column as an extraction device. The extracted TSNAs were easily desorbed from the column by passage of the mobile phase, with no carryover observed. This in-tube SPME LC-MS/MS method showed good linearity for four TSNAs in the 0.5-100 pg mL-1 ranges, with correlation coefficients above 0.9998 (n = 24), using stable isotope-labeled TSNAs as internal standards. The validation assays for limit of detection, limit of quantification, specificity, precision and accuracy of the analytes were consistent with the requirements recommended by the ICH guidelines. The validated method was utilized successfully to analyze four TSNAs in mainstream and sidestream smoke samples without any interference peaks. Overall recoveries of TSNAs spiked into smoke sample solutions were 97.3-104.6%. The developed method can automate the extraction, concentration and analysis of samples, and is sensitive and selective for TSNAs. This method was used to analyze TSNAs in mainstream and sidestream smoke samples of several commercially available combustion cigarettes and heated tobacco products.

Development of Ion Mobility Spectrometry with Novel Atmospheric Electron Emission Ionization for Field Detection of Gaseous and Blister Chemical Warfare Agents.

Drift tube ion mobility spectrometry with a novel atmospheric electron emission (AEE) source was developed for determination of gaseous and blister chemical warfare agents (CWAs) in negative mode. The AEE source was fabricated from an aluminum substrate electrode covered with 1 µm silver nanoparticle-dispersed silicone resin and a thin gold layer. This structure enabled stable tunneling electron emission upon the application of more than 11 V potential under atmospheric pressure. The reactant ion peak (RIP) was observed for the reduced mobility constant ( K0) of 2.18 and optimized at the charging voltage of 20 V. This RIP was assigned to O2- by using a mass spectrometer. Hydrogen cyanide was detected as a peak ( K0 = 2.47) that was discriminatively separated from the RIP (resolution = 1.4), with a limit of detection (LOD) of 0.057 mg/m3, and assigned to CN- and OCN-. Phosgene was detected as a peak ( K0 = 2.36; resolution = 1.2; and LOD = 0.6 mg/m3), which was assigned to Cl-. Lewisite 1 was detected as two peaks ( K0 = 1.68 and 1.34; LOD = 12 and 15 mg/m3). The K0 = 1.68 peak was ascribed to a mixture of adducts of molecules or the product of hydrolysis with oxygen or chloride. Cyanogen chloride, chlorine, and sulfur mustard were also well detected. The detection performance with the AEE source was compared with those under corona discharge and 63Ni ionizations. The advantage of the AEE source is the simple RIP pattern (only O2-), and the characteristic marker ions contribute to the discriminative CWAs detection.

Fundamental study of radiogallium-labeled aspartic acid peptides introducing octreotate derivatives.

OBJECTIVE: Somatostatin receptors are highly expressed in neuroendocrine tumors, and many radiolabeled somatostatin analogs for diagnosis and treatment have been developed. To simultaneously detect not only primary cancer but also bone metastases, this study aimed to develop a positron emission tomography probe using generator-produced nuclide Gallium-68 (T1/2 = 68 min), in which a carrier for primary cancer, a carrier for bone metastases lesions, and a stable gallium complex are introduced into the one molecule. Based on this strategy, the somatostatin receptor-targeted peptide, [Tyr3]-octreotate (TATE), aspartic acid peptide (Dn) with high binding affinity for hydroxyapatite, and Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a stable gallium complex were selected. The novel complexes, Ga-DOTA-Dn-TATE (n = 0, 2, 5, 8, or 11), were designed, synthesized, and evaluated. The radiogallium complexes were prepared using the easy-to-handle radioisotope 67Ga due to relatively long half-life. METHODS: The radiogallium complex precursor DOTA-Dn-TATE was synthesized by the Fmoc-based solid-phase method and by the air oxidation method to form the disulfide bond. [67Ga]Ga-DOTA-Dn-TATE was synthesized by reacting DOTA-Dn-TATE and 67Ga. Hydroxyapatite binding assays, in vitro cellular uptake experiments in AR42J tumor cells, in biodistribution experiments in AR42J tumor-bearing mice, were performed using [67Ga]Ga-DOTA-Dn-TATE. RESULTS: The radiochemical purities of [67Ga]Ga-DOTA-Dn-TATE were > 96.0%. In in vitro and in vivo experiments, [67Ga]Ga-DOTA-D11-TATE had a high affinity for hydroxyapatite and highly accumulated in bone. However, the uptake of [67Ga]Ga-DOTA-D11-TATE into somatostatin receptor-positive AR42J cells was lower than that of [67Ga]Ga-DOTA-TATE, and the accumulation of [67Ga]Ga-DOTA-D11-TATE in tumor was significantly low. CONCLUSION: Ga-DOTA-D11-TATE may not be recognized by somatostatin receptor by the introduction of D11, and the charge adjustment may be important for somatostatin receptor-positive cell uptake.

Evaluation of Ga-DOTA-(D-Asp)n as bone imaging agents: D-aspartic acid peptides as carriers to bone.

67Ga-DOTA-(L-Asp)11 and 67Ga-DOTA-(L-Asp)14, which have been developed as bone imaging agents, showed a high accumulation in bone and a rapid blood clearance in mice. However, peptides composed of D-amino acids are more stable in vivo than those composed of their L-equivalents. In this study, 67Ga-DOTA-(D-Asp)n (n = 2, 5, 8, 11, or 14) were synthesized using the Fmoc-based solid-phase methodology and evaluated. In hydroxyapatite binding assay, binding of 67Ga-DOTA-(D-Asp)n tended to increase with increasing length of the amino acid chain. 67Ga-DOTA-(D-Asp)11 and 67Ga-DOTA-(D-Asp)14 caused a high accumulation of radioactivity in the bones of the mice. However, the results for 67Ga-DOTA-(D-Asp)n and 67Ga-DOTA-(L-Asp)n were comparable. In urine analyses, the proportion of intact complex after injection of 67Ga-DOTA-(D-Asp)14 was significantly higher than that of 67Ga-DOTA-(L-Asp)14. Although 67Ga-DOTA-(D-Asp)14 was more stable than 67Ga-DOTA-(L-Asp)14, the properties of 67Ga-DOTA-(D-Asp)n and 67Ga-DOTA-(L-Asp)n as bone imaging agents may be comparable.

Characterization of Catechins in Water by Photoemission Yield Spectroscopy in Air.

Photoemission yield spectroscopy in air (PYSA) was applied for the characterization of catechins in water in ambient conditions. According to the results of measurements on aqueous solutions of epigallocatechin gallate (EGCg) of various concentrations, the photoemission yield is almost proportional to the concentration of EGCg. Contrarily, the threshold energy of photoemission, EPET, is almost constant at 5.46 ± 0.02 eV. Moreover, we measured aqueous solutions of epicatechin (EC), epigallocatechin (EGC), and epicatechin gallate (ECg). The values of EPET of EC, EGC, ECg were estimated to be 5.72 ± 0.02, 5.68 ± 0.01, and 5.45 ± 0.02 eV, respectively, and a dependence on the molecular structure was found. Furthermore, changes in the photoemission yield spectra of heated EGCg were well explained by molecular orbital calculations on the basis of an assumption of epimerization.

Biomonitoring method for the determination of polycyclic aromatic hydrocarbons in hair by online in-tube solid-phase microextraction coupled with high performance liquid chromatography and fluorescence detection.

Polycyclic aromatic hydrocarbons (PAHs) are formed from the incomplete combustion or pyrolysis of organic matter during industrial processing and various human activities, but human exposure to PAHs has not yet been elucidated in detail. To assess long-term exposure to PAHs, we developed a simple and sensitive method for measuring PAHs in hair by online in-tube solid-phase microextraction using a CP-Sil 19CB capillary column as an extraction device, followed by high-performance liquid chromatography using a Zorbax Eclipse PAH column and fluorescence detection. Seventeen PAHs could be analyzed simultaneously, with good linearity from 20 to 1000pg/mL each as determined using stable isotope-labeled PAH internal standards. The detection limits of PAHs were 0.5-20.4pg/mL. PAHs in human hair samples were extracted by ultrasonication in 50mM NaOH in methanol, and successfully analyzed without any interference peaks, with good recovery rates above 70% in spiked hair samples. Using this method, we evaluated the suitability of using hair PAHs as biomarkers for long-term exposure.

Well-designed bone-seeking radiolabeled compounds for diagnosis and therapy of bone metastases.

Bone-seeking radiopharmaceuticals are frequently used as diagnostic agents in nuclear medicine, because they can detect bone disorders before anatomical changes occur. Furthermore, their effectiveness in the palliation of metastatic bone cancer pain has been demonstrated in the clinical setting. With the aim of developing superior bone-seeking radiopharmaceuticals, many compounds have been designed, prepared, and evaluated. Here, several well-designed bone-seeking compounds used for diagnostic and therapeutic use, having the concept of radiometal complexes conjugated to carrier molecules to bone, are reviewed.

Radiogallium Complex-Conjugated Bifunctional Peptides for Detecting Primary Cancer and Bone Metastases Simultaneously.

(68)Ga (T(1/2) = 68 min, a generator-produced nuclide) is an interesting radionuclide for clinical positron emission tomography (PET). Recently, it was reported that radiogallium-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated (Asp)n peptide [Ga-DOTA-(Asp)n] has great potential for bone metastases imaging. In the current study, a compound containing an aspartic acid peptide linker (D11) as a carrier to bone metastases, an RGD peptide [c(RGDfK) peptide] as a carrier to the primary cancer, and Ga-DOTA as a stable radiometal complex for imaging in one molecule, Ga-DOTA-D11-c(RGDfK), was designed, prepared, and evaluated to detect both the primary cancer and bone metastases simultaneously using (67)Ga, which is easy to handle. After DOTA-D11-c(RGDfK) was synthesized using Fmoc-based solid-phase methodology, (67)Ga-DOTA-D11-c(RGDfK) was prepared by complexing DOTA-D11-c(RGDfK) with (67)Ga. Hydroxyapatite binding assays, integrin binding assays, biodistribution experiments, and single photon emission tomography (SPECT) imaging using tumor-bearing mice were performed using (67)Ga-DOTA-D11-c(RGDfK). (67)Ga-DOTA-D11-c(RGDfK) was prepared with a radiochemical purity of >97%. In vitro, (67)Ga-DOTA-D11-c(RGDfK) had a high affinity for hydroxyapatite and αvß3 integrin. In vivo, (67)Ga-DOTA-D11-c(RGDfK) exhibited high uptake in bone and tumor. The accumulation of (67)Ga-DOTA-D11-c(RGDfK) in tumor decreased when it was co-injected with c(RGDfK) peptide. (68)Ga-DOTA-D11-c(RGDfK) has great potential as a PET tracer for the diagnosis of both the primary cancer and bone metastases simultaneously.

In situ measurements of the desorption of water from a TiO2 surface under dry air by collecting the photoemission yield with an open counter.

We investigated the desorption of water from a TiO2 surface under a dry atmosphere by collecting the photoemission yield spectra with an open counter. For this purpose, a new attachment for the photoemission yield measurement was prepared. This apparatus is capable of detecting, in the open air, low-energy electrons excited by photons under dried atmospheres; the dew point is below -35°C. A significant change in the photoemission yield spectra due to exposure to a dry atmosphere was observed. To gain a better understanding of these results, observations of the change in the photoemission yield spectra caused by the thermal desorption of adsorbed water were also carried out. The results are consistent with those obtained by exposure to a dry atmosphere. Based on the relationship between the photoemission yield and the thickness of the water layer, the time dependence of the change in the thickness was explained by the second-order reaction rate equation.

Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

(68)Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68)Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle (67)Ga, with the previously described (67)Ga-DOTA complex conjugated bisphosphonate, (67)Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with (67)Ga, resulting in (67)Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67)Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67)Ga-DOTA-(Asp)8, (67)Ga-DOTA-(Asp)11, and (67)Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67)Ga-DOTA-(Asp)n was lower than that of (67)Ga-DOTA-Bn-SCN-HBP, blood clearance of (67)Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of (67)Ga-DOTA-(Asp)11 and (67)Ga-DOTA-(Asp)14 were comparable with those of (67)Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68)Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

Analysis of contaminant polycyclic aromatic hydrocarbons in tea products and crude drugs.

Tea products and crude drugs were analyzed to determine levels of contamination with 15 polycyclic aromatic hydrocarbons (PAHs) using a new online automated method, consisting of in-tube solid-phase microextraction (SPME) coupled with high-performance liquid chromatography-fluorescence detection (HPLC-FLD). PAHs were separated within 15 min by HPLC using a Zorbax Eclipse PAH column with a water/acetonitrile gradient elution program as the mobile phase. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 µL of sample using a CP-Sil 19CB capillary column as an extraction device. Low- and high-molecular weight PAHs were extracted effectively onto the capillary coating from 5% and 30% methanol solutions, respectively. The extracted PAHs were readily desorbed from the capillary by passage of the mobile phase. Good linearity of the calibration curve (r > 0.9972) was obtained in the concentration range of 0.05-2.0 ng mL-1, and the detection limits (S/N = 3) of PAHs were 0.32-4.63 pg mL-1. Using the in-tube SPME/HPLC-FLD method, PAHs were detected at ng g-1 levels in the samples without interference peaks. Our results suggest that PAHs contamination in herbal products is widespread, and the proposed method may become a useful tool for monitoring PAH contamination and quality control in herbal products.

Determination of anabolic steroids in human urine by automated in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

A simple, rapid and sensitive method was developed for determining the presence of seven anabolic steroids (boldenone, nandrolone, testosterone, methyltestosterone, epiandrosterone, androsterone, and atnozolol) in human urine. Glucuronide-conjugates of these compounds were hydrolyzed with beta-glucuronidase. The anabolic steroids were analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). The steroids were separated within 14 min by high performance liquid chromatography using a Chromolith RP-18e column and 5 mM ammonium formate/methanol (35/65, v/v) as a mobile phase at a flow rate of 1.0 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for the MS detection of these compounds. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 microL using a Supel-Q PLOT capillary column for the extraction. The extracted compounds could be desorbed readily from the capillary column by flow of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS with SIM mode detection, good linearity of the calibration curve (r>0.995) was obtained in the concentration range of 0.5-20 ng/mL, except for stanozolol. The detection limits (S/N=3) of anabolic steroids were in the range 9-182 pg/mL and the proposed method showed 20-33-fold higher sensitivity than the direct injection method. The within-day and between-day precisions were below 4.0% and 7.3% (n=5), respectively. This method was applied successfully to the analysis of urine samples without the interference peaks. The recovery rates of anabolic steroids spiked into urine samples were above 85%. This method is useful to analyze the urinary levels of these compounds in anti-doping tests.

Determination of perfluorooctanoic acid and perfluorooctane sulfonate by automated in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

We have developed a simple, rapid, and sensitive method for the determination of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). PFOA and PFOS were separated within 10 min by high-performance liquid chromatography using an Inertsil ODS-3 column and 10 mM ammonium acetate/methanol (35/65, v/v) as a mobile phase at a flow rate of 0.25 mL min(-1). Electrospray ionization conditions in the negative ion mode were optimized for MS detection of PFOA and PFOS. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 microL using a CP-Pora PLOT amine capillary column as the extraction device. The extracted compounds could be desorbed easily from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS method, good linearity of the calibration curve (r=0.9990 for PFOA, r=0.9982 for PFOS) was obtained in the range of 0.05-5 ng mL(-1) each compound. The detection limits (S/N=3) for PFOA and PFOS were 1.5 and 3.2 pg mL(-1), respectively. The method described here showed about 100-fold higher sensitivity than the direct injection method. The within-day and between-day precisions (relative standard deviations) were below 3.7 and 6.0%, respectively. This method was applied successfully to the analysis of PFOA and PFOS in environmental water samples and to the elution test from a Teflon-coated frying pan without interference peaks. The recoveries of PFOA and PFOS spiked into river samples were above 81%, and PFOA was detected at pg mL(-1) levels in environmental water samples and eluate from the frying pan.

Developments and applications of capillary microextraction techniques: a review.

Sample preparation is important for isolating desired components from complex matrices and greatly influences their reliable and accurate analysis. Recent trends in sample preparation include miniaturization, automation, high-throughput performance, and reduction in solvent consumption and operation time. This review focuses on novel microextraction techniques using capillaries for off-line and on-line sample preparation. Open-tubular trapping (OTT), in-tube solid-phase microextraction (SPME), wire-in-tube SPME, fiber-in-tube solid-phase extraction (SPE), sorbent-packed capillary in-tube SPME and monolithic capillary in-tube SPME are critically evaluated and applications of these techniques in biological, pharmaceutical, environmental and food analyses are summarized.