Prior Clean-up Research Articles

A simple method for the selective quantification of polyethylene, polypropylene, and polystyrene plastic debris in soil by pyrolysis-gas chromatography/mass spectrometry

The lack of adequate analytical methods for the quantification of plastic debris in soil challenges a better understanding of their occurrence and fate in the terrestrial environment. With this proof-of-principle study, we developed a simple and fast method for the selective quantification of the three most environmentally relevant polymers polyethylene (PE), polypropylene (PP), and polystyrene (PS) in soil using pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). In order to facilitate the preparation of calibration series and to better account for the heterogeneity of soil matrix, polymers were dissolved in 1,2,4-trichlorobenzene (TCB) at 120 °C. Thereby, liquid sample aliquots from up to 4 g of solid sample became amenable to pyrolysis without further preparation. To evaluate the performance of this approach, three reference soils with 1.73–5.16% organic carbon (Corg) were spiked at 50 and 250 μg g−1 of each polymer and extracted with TCB. Prior cleanup steps with methanol, flocculation with KAl(SO4)2, or Fenton digestion were tested for their suitability to reduce potentially interfering Corg. Calibration curves responded linearly (adj. R2 > 0.996) with instrumental detection limits of 1–86 ng corresponding to method detection limits of 1–86 μg g−1. The measurement repeatability was 3.2–7.2% relative standard deviation. Recoveries of 70–128% were achieved for plastic contents of 250 μg g−1 extracted with TCB without prior cleanup from soils with less than 2.5% Corg. A higher Corg particularly interfered with the quantification of PE. The addition of non-target polymers (polyethylene terephthalate, polyvinyl chloride, poly(methyl methacrylate), and tire wear particles) did not interfere with the quantification of the analytes highlighting the selectivity of the method. Further research is needed to determine low plastic contents in soils exceeding 2.5% Corg. With 1–3 h processing time per sample, our method has the potential for routine analyses and screening studies of agricultural systems to be complemented with microspectroscopic techniques for additional information on particle shapes and sizes.

Sensitive determination of plasma protein binding of cationic drugs using mixed-mode solid-phase microextraction

Freely dissolved concentrations are considered to be the most relevant concentration in pharmacology and toxicology, as they represent the active concentration available for interaction with its surroundings. Here, a solid-phase microextraction (SPME) coating that combines octadecyl and propylsulfonic acid groups as strong cation exchange sites, known as C18/SCX or “mixed-mode” SPME, is used to measure freely dissolved concentrations of amitriptyline, amphetamine, diazepam and tramadol to different binding matrices, including bovine serum albumin (BSA), human serum albumin (HSA), human plasma and human whole blood. A potential confounding factor in binding studies is that proteins may sorb to the fiber coating leading to incorrect measurement of protein sorption or changes in uptake kinetics to the fiber coating. Sorption of bovine serum albumin (BSA) was observed and quantified using a Lowry assay. BSA binds to the C18/SCX fiber in small amounts, but large changes in uptake kinetics were not observed. All experiments were performed at equilibrium. In addition, however, the effect of depletion and non-equilibrium extraction on the estimation of protein binding affinities was also studied. Binding affinities to BSA and human serum albumin (HSA) were calculated as log KBSA or log KHSA. These values were very similar to reported literature values. Sampling at either equilibrium or non-equilibrium resulted in similar binding affinities. Furthermore, SPME fibers were used to measure freely dissolved concentrations in undiluted human plasma and whole blood. Analysis of SPME extracts could be performed using HPLC-UV or HPLC with fluorescence detection without prior clean-up of the samples. Measured bound fractions in plasma using this SPME approach were comparable to literature reference values. Bound fractions in whole blood were always higher than in plasma, due to red blood cell partitioning. This work shows the potential of SPME as sampling tool for freely dissolved concentrations, especially for highly protein-bound compounds. Conventional SPME coatings such as polyacrylate (PA) or polydimethylsiloxane (PDMS) might be lacking sensitivity when sampling the small neutral fraction of highly protein-bound positively charged compounds, but the C18/SCX fiber is able to sorb the charged species of organic cations, thereby improving sensitivity for these types of compounds.

Isolation and Fast Selective Determination of Nor-abietanoid Diterpenoids from Perovskia atriplicifolia Roots Using LC-ESI-MS/MS with Multiple Reaction Monitoring

In the first part of this study we extracted, isolated, and identified the main diterpenoid constituents from the roots of a Central Asian medicinal and ornamental plant--Perovskia atriplicifolia Benth. Eight major nor-abietanoid pigments were obtained using NP silica gel column chromatography and preparative RP-HPLC: cryptotanshinone, 1-hydroxycryptotanhinone, miltirone, 1-oxomiltirone, tanshinone IIa, 1,2 didehydrotanshinone IIa, 1,2 didehydromiltirone, the non-quinone diterpenoid - arucadiol, as well as rosmarinic acid as a main phenolic compound. Secondly, we used the obtained compounds for fast and selective determination of the main diterpenes present in P. atriplicifolia root extract. After extraction with n-hexane, the quantitative analysis was carried out by LC-MS/MS with a triple quadrupole (qQq) mass detector without any prior clean-up step. Identification of the diterpenes was confirmed by multiple reaction monitoring (MRM) using the most representative transitions from the precursor ions, while the most sensitive transitions were used for quantification in a 19-minute run. Most of the isolated and analyzed compounds had not been previously reported from this species. This easily cultivated plant is a promising source of several pharmacologically valuable abietanoid diterpenoids.

Influence of deep-fat frying process on phospholipid molecular species composition of Sardina pilchardus fillet

The effects of deep-fat frying performed using different culinary fats (extra virgin olive oil, conventional sunflower oil and high-oleic sunflower oil) and different frying temperatures (160 and 180 °C) on the composition of the preponderant fish phospholipid classes, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), were investigated on Sardina pilchardus. The total fish lipid fraction was injected into the HPLC system coupled on line with a second order mass spectrometer (MS–MS) by means of electronebulization interface (ESI), without a prior clean-up of the phospholipid fraction.The deep-fat frying process caused significant changes on PE and PC molecular species composition of the fish fillet.In all cases, the deep-fat frying process caused a significant (P < 0.05) increase in the relative proportions of the PE and PC species constituted by the combination of palmitic and docosahexaenoic acids. At the same time, a depletion of the percentage of the PE and PC species formed by two docosahexaenoic acid residues in fried fillets was registered. Anyway, this depletion was statistically significant for PC, but not for PE.Both PE and PC compositions were not influenced by the frying temperature, whereas the nature of the culinary fat had an effect on the PC composition. Particularly, the frying tests with conventional sunflower oil produced a statistically significant increase of PC species containing saturated/polyunsaturated fatty acids and a significant decrease of PC species formed by the combination of two polyunsaturated fatty acids.

Novel one-step headspace dynamic in-syringe liquid phase derivatization–extraction technique for the determination of aqueous aliphatic amines by liquid chromatography with fluorescence detection

A novel one-step headspace (HS) dynamic in-syringe (DIS) based liquid-phase derivatization–extraction (LPDE) technique has been developed for the selective determination of two short-chain aliphatic amines (SCAAs) in aqueous samples using high performance liquid chromatography (HPLC) with fluorescence detection (FLD). Methylamine (MA) and dimethylamine (DMA) were selected as model compounds of SCAAs. In this method, a micro-syringe pre-filled with derivatizing reagent solution (9-fluorenylmethyl chloroformate) in the barrel was applied to achieve the simultaneous derivatization and extraction of two methylamines evolved from alkalized aqueous samples through the automated reciprocated movements of syringe plunger. After the derivatization–extraction process, the derivatized phase was directly injected into HPLC-FLD for analysis. Parameters influencing the evolution of methylamines and the HS-DIS-LPDE efficiency, including sample pH and temperature, sampling time, as well as the composition of derivatization reagent, reaction temperature, and frequency of reciprocated plunger movements, were thoroughly examined and optimized. Under optimal conditions, detections were linear in the range of 25–500μgL−1 for MA and DMA with correlation coefficients all above 0.995. The limits of detection (based on S/N=3) were 5 and 19ngmL−1 for MA and DMA, respectively. The applicability of the developed method was demonstrated for the determination of MA and DMA in real water samples without any prior cleanup of the sample. The present method provides a simple, selective, automated, low cost and eco-friendly procedure to determine aliphatic amines in aqueous samples.

Co-occurrence and statistical correlations between mycotoxins in feedstuffs collected in the Asia–Oceania in 2010

In this study, a total of 1468 feed samples collected in the Asian-Oceania region during the year 2010 were analysed for the presence of aflatoxins B1, B2, G1 and G2, fumonisin B1 and B2, zearalenone, ochratoxin A and deoxynivalenol. The samples tested were diverse, ranging from cereals such as corn, wheat and rice to processing by-products, namely soybean meal, corn gluten meal, dried distillers grains with solubles and other fodder, including straw, silage and finished feed. The samples were analysed by using high liquid chromatography with UV detector, postcolumn fluorescence derivatization and mass spectrometer after prior clean-up. The positive correlation between the occurrence of aflatoxins B1 and B2 and fumonisins B1 and B2 was very high, as was to be expected. Both correlation between the Fusarium mycotoxins deoxynivalenol and zearalenone and the storage mycotoxins ochratoxin A and aflatoxins proved to be low. The correlations between individual mycotoxins depended strongly on the commodity itself. These data indicate that the contamination of feedstuffs with only one mycotoxin is rare and that mycotoxins occur more frequently together, representing a risk for domestic livestock.

Determination of ammonium in aqueous samples using new headspace dynamic in-syringe liquid-phase microextraction with in situ derivitazation coupled with liquid chromatography–fluorescence detection

A new simultaneous derivatization and extraction method for the preconcentration of ammonia using new one-step headspace dynamic in-syringe liquid-phase microextraction with in situ derivatization was developed for the trace determination of ammonium in aqueous samples by liquid chromatography with fluorescence detection (LC–FLD). The acceptor phase (as derivatization reagent) containing o-phthaldehyde and sodium sulfite was held within a syringe barrel and immersed in the headspace of sample container. The gaseous ammonia from the alkalized aqueous sample formed a stable isoindole derivative with the acceptor phase inside the syringe barrel through the reciprocated movements of plunger. After derivatization-cum-extraction, the acceptor phase was directly injected into LC–FLD for analysis. Parameters affecting the ammonia evolution and the extraction/derivatization efficiency such as sample matrix, pH, temperature, sampling time, and the composition of derivatization reagent, reaction temperature, and frequency of reciprocated plunger, were studied thoroughly. Results indicated that the maximum extraction efficiency was obtained by using 100μL derivatization reagent in a 1-mL gastight syringe under 8 reciprocated movements of plunger per min to extract ammonia evolved from a 20mL alkalized aqueous solution at 70°C (preheated 4min) with 380rpm stirring for 8min. The detection was linear in the concentration range of 0.625–10μM with the correlation coefficient of 0.9967 and detection limit of 0.33μM (5.6ngmL−1) based on SN−1=3. The method was applied successfully to determine ammonium in real water samples without any prior cleanup of the samples, and has been proved to be a simple, sensitive, efficient and cost-effective procedure for trace ammonium determination in aqueous samples.

Development and validation of a rapid multi‐biomarker liquid chromatography/tandem mass spectrometry method to assess human exposure to mycotoxins

Mycotoxins regularly occur in food worldwide and pose serious health risks to consumers. Since individuals can be exposed to a variety of these toxic secondary metabolites of fungi at the same time, there is a demand for proper analytical methods to assess human exposure by suitable biomarkers. This study reports on the development of a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative measurement of 15 mycotoxins and key metabolites in human urine using polarity switching. Deoxynivalenol (DON), DON-3-O-glucuronide, DON-15-O-glucuronide (D15GlcA), de-epoxy DON, nivalenol (NIV), T-2 toxin, HT-2 toxin, zearalenone, zearalenone-14-O-glucuronide, α- and β-zearalenol, fumonisins B(1) and B(2) (FB(1), FB(2)), ochratoxin A (OTA) and aflatoxin M(1) (AFM(1)) were determined without the need for any cleanup using a rapid and simple dilute and shoot approach. Validation was performed in the range of 0.005-40 µg L(-1) depending on the analyte and expected urinary concentration levels. Apparent recoveries between 78 and 119% and interday precisions of 2-17% relative standard deviation (RSD) were achieved. The applicability of the method was demonstrated by the analysis of urine samples obtained from Cameroon. In naturally contaminated urine samples up to six biomarkers of exposure (AFM(1), DON, D15GlcA, NIV, FB(1), and OTA) were detected simultaneously. We conclude that the developed LC/MS/MS method is well suited to quantify multiple mycotoxin biomarkers in human urine down to the sub-ppb range within 18 min and without any prior cleanup. The co-occurrence of several mycotoxins in the investigated samples clearly emphasizes the great potential and importance of this method to assess exposure of humans and animals to naturally occurring mycotoxins.

A VALIDATED HIGH-THROUGHPUT CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS DETERMINATION OF VITAMIN K HOMOLOGUES

Vitamin K homologues are highly lipophilic compounds that require long separation times on chromatographic analysis which does not meet the demand of higher sample throughputs in quality control laboratories. Therefore, this study aimed to develop a new validated high-throughput high-performance thin-layer chromatographic (HPTLC) method to quantify vitamin K homologues including phylloquinone (PK, vitamin K1), menaquinone-4 (MK-4, vitamin K2), and menaquinone-7 (MK-7, vitamin K2). The densitometric analysis was carried out using HPTLC silica gel G 60 F254 plates as the stationary phase. The plates were developed with methanol-ethanol-isopropanol-water (75:5:5:15, v/v/v/v) in the absorbance mode at 254 nm. The retention factors of MK-4, PK, and MK-7 were 0.56, 0.43, and 0.23, respectively. Linearity was found to be in the range of 1–200 ng band−1 for PK and MK-4 and 2–200 ng band−1 for MK-7 with correlation coefficient of 0.9990 or more. The limits of detection and quantitation were 0.19–0.85 and 0.76–2.5 ng band−1, respectively. The method was validated in accordance ICH guidelines. The method was applied for determination of vitamin K homologues in pharmaceutical formulations and food samples after extraction without prior clean-up procedures. The developed HPTLC method provides a useful tool for rapid and efficient high-throughput analysis of vitamin K homologues.

Aflatoxin M&lt;sub&gt;1&lt;/sub&gt; in bulk-tank raw milk produced in a low risk area

The present study was designed to determine the levels of aflatoxin M 1 (AFM 1 ) and its seasonal variation in bulk tank cow milk collected from dairy farms located on the Italian island of Sardinia. Cow bulk tank milk samples (No 356) were collected from 178 farms twice a year, during the winter and summer seasons. All samples were analysed for AFM 1 with a commercial ELISA kit. Confirmation of AFM 1 positive samples (>10 ng L -1 ) was accomplished by using high-performance liquid chromatography (HPLC) with a prior clean-up step using immunoaffinity columns. AFM 1 was found at levels ≥5 ng L -1 in 112 samples (31.5%) but only one exceeded the maximum EC level (50 ng L -1 ). There was no seasonal influence on the aflatoxin content of the milk samples analysed.

Variation of levels of aflatoxin M 1 in raw milk from different localities in the central areas of Punjab, Pakistan

The occurrence of aflatoxin M 1 (AFM 1) in samples of raw milk of buffaloes and cows from different localities in the central areas of Punjab, the province of Pakistan, was determined by using high-performance liquid chromatography (HPLC) with prior clean-up step applying immunoaffinity columns. The present study has been designed to find out the variation of levels of aflatoxin M 1 in raw milk of different localities. Feed has major role for prevalence of aflatoxin M 1 in milk and different feed regimen is being used in different areas. Total 480 milk samples were analyzed, among these 360 were of buffalo milk and 120 were of cow milk. The percentage of AFM 1 contamination in buffalo milk and cow milk was 42.5% and 52.5%, respectively. The mean value of AFM 1 was 0.027 μg L −1 in buffaloes’ milk and was 0.044 μg L −1 in cows’ milk. In both types of milk, level of AFM 1 concentration was higher in milk samples obtained from urban and semi-urban areas and it was minimal in milk from rural areas.

Quantitative analysis of Tenecteplase in rat plasma samples using LC–MS/MS as an alternative for ELISA

An LC–MS/MS method has been developed for the quantitative determination of a protein drug (Tenecteplase; M W 58,777 Da) in rat plasma. The protein was digested with trypsin without prior clean-up of the plasma sample, without the use of a label nor internal standard. A limited validation was performed to assess the linearity, the sensitivity and the specificity of the method. In addition, the developed method was applied to the quantitative analysis of Tenecteplase in rat plasma samples originating from a single-dose study in rats.

Validation of two analytical methods for the determination of ochratoxin A by reversed-phased high-performance liquid chromatography coupled to fluorescence detection in musts and sweet wines from Andalusia

Some Spanish sweet wines are made from raisins, grapes dried by direct exposure to the sun after picking. This drying process can encourage ochratoxin A (OTA) formation. OTA is a mycotoxin formed by several fungi. It has been linked to nephropathy in humans, and may have a long half-life in humans. The aim of this study is to develop and to apply two procedures for the analysis of OTA in grape musts (during the raisining process) and sweet wines, respectively. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to fluorescence detection (FLD) was employed in both analytical methods. In grape must, the method involves the direct injection of the sample in a HPLC-FLD system without any kind of prior clean-up procedure. The complexity of the sweet wine samples requires a solid-phase extraction (SPE) clean-up on a C18 column which enables the OTA to be isolated from the matrix. The methods used were statistically validated. The validation also included the comparison of the slopes of the curve obtained with standards and the regression curves obtained by the addition of a standard. Two different studies of standard additions were conducted. One method was validated without sample preparation and it was applied to must samples. The other method was validated with SPE extraction and it was applied to sweet wine samples. Recovery was always better than 89.69%. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were established at 0.22 and 0.77 μg l −1, respectively. In general, the analytical data obtained provided good results at the sub-μg l −1 concentration level.

Electrospray mass spectrometry to study lake water DOM and effects of microbial degradation

Qualitative analysis of lake water dissolved organic matter (DOM) was carried out using electrospray ionization mass spectrometry (ESI MS) without prior clean-up or chromatography, showing the potential of ESI MS as a rapid method to analyse qualitative differences between DOM with different environmental properties. Spectra were evaluated in terms of the relative ion abundance in certain mass to charge ranges (m/z range), and the mass-to-charge average (m/z average). Lake water DOM from two different sources was used. In addition, DOM from both sources that had been subjected to microbial degradation under different oxygen regimes was analysed. ESI MS results were compared with those of UV absorbance and pollutant binding capacity (pyrene and atrazine). The results from all methods indicated qualitative differences between the DOM from different lakes, but only minor qualitative changes when comparing oxic and anoxic degradation. To detect those small differences the m/z range proved to be the more sensitive evaluation method, compared to the m/z average.

2-Alkylcyclobutanones as Irradiation Dose Indicators in Irradiated Ground Beef Patties

Alkylcyclobutanones have been recognized as chemical markers of irradiated lipid-containing foods since 1970. They are important because they are produced solely as a result of irradiation and not any other processing method. This study investigated the formation of 2-dodecylcyclobutanone (2-DCB) and 2-tetradec-5'-enylcyclobutanone (2-TDCB) in irradiated ground beef patties from commercial and noncommercial sources. Patties were irradiated using a (60)C source (gamma-irradiation) and electron beam irradiation, at five targeted absorbed doses of 0.5, 1.0, 2.5, 5.0, and 7.0 kGy. Commercially available irradiated patties were also studied. A supercritical fluid extraction (SFE) procedure was optimized and used for the extraction and isolation of the alkylcyclobutanones. Samples can be used for extraction without a prior cleanup step, which makes this procedure rapid and convenient to use. Identification and quantitation of the cyclobutanones were done by gas chromatography-mass spectroscopy. 2-DCB was detected in all of the irradiated samples (including commercial patties), and its concentration increased linearly with the irradiation dose. Electron beam irradiation produced a greater amount of 2-DCB compared to gamma-irradiation at dose levels >2.5 kGy. 2-TDCB was detected only at the two higher irradiation doses, whereas both marker compounds were not detected in the non-irradiated samples.

Liquid Chromatographic Detection of Dowtherm A Contamination in Oleochemicals and Edible Oils

A rapid and direct liquid chromatographic (LC) technique is described for the determination of a eutectic mixture of diphenyl oxide and biphenyl such as Dowtherm A thermal heating fluid (THF) in oleochemicals and palm olein. Analysis is performed with an RP-18 column with fluorescence detection (excitation at 247 nm and emission at 310 nm). The isocratic mobile phase (1.0 mL/min) consists of methanol and water (90 + 10, v/v). A linear calibration model (correlation coefficient = 0.9999) was developed directly from used Dowtherm A THF with the biphenyl peak (4.70 min) as a marker. Average recoveries from spiked glycerin, fatty alcohol mixture, methyl ester mixture, fatty acids, and palm olein were 90.9-108.7%, with a detection limit of 0.1 microgram/mL. The technique requires no prior sample cleanup nor extraction steps and is good for quality assurance purposes.

Sensitive and specific method for the simultaneous determination of natural and synthetic catecholamines and 3,4-dihydroxyphenylglycol in microdialysis samples

The relatively new technique of microdialysis provides new possibilities for investigating in vivo the functioning of the sympathetic nervous system. The small sample volumes obtained, however, are a great challenge for analytical chemists. We report here a HPLC method for measuring in one run both natural and synthetic catecholamines [dopamine, (nor)epinephrine, α-methylnorepinephrine, isoproterenol and epinine] and the intraneuronal metabolite 3,4-dihydroxyphenylglycol in small microdialysis samples after derivatization with the fluorogenic agent 1,2-diphenylethylenediamine. No prior clean-up step is necessary. N-Ethylmaleimide is necessary for preventing an inhibitory action on derivatization occurring in in vivo microdialysis samples. The method can handle large numbers of samples, is sensitive (on-column detection limits 30 to 200 fg) and reproducible (RSD 1 to 7%). Recovery characteristics of the commercial microdialysis probe used (CMA/20) were extensively investigated both in vitro and in vivo at various perfusion rates; for practical purposes a rate of 2 μl/min and sampling at 10-min intervals was found to be workable and to give good and reproducible recoveries (50 to 70%).

Determination of chemical warfare agent degradation products at low-part-per-billion levels in aqueous samples and sub-part-per-million levels in soils using capillary electrophoresis.

A significant enhancement in the method detection limits is observed in the analysis of chemical warfare agent (CWA) degradation products in environmental samples by capillary electrophoresis (CE) using electrokinetic injection. The CE method uses indirect UV detection of the nonderivitized acidic analyte and a cationic surfactant, didodecyldimethylammonium hydroxide, for reversal of the electroosmotic flow. Analytes studied include the dibasic acid methylphosphonic acid (MPA) and its monoacid/monoalkyl esters, RMPA, where R = ethyl, isopropyl, and pinacolyl (2-(3,3-dimethylbutyl)). The CE method uses an attractive buffer system which is highly stable and inexpensive, and, in addition to reversing the electroosmotic flow, provides excellent separation efficiencies within a 3-min run. This CE method is also free from interference caused by carbonate, humic acids, and fluoride. Compared to pressure injection, electrokinetic injection with this CE buffer system provided substantially lower detection limits, up to 100-fold lower for samples in reagent water. However, to best realize the benefits of the electrokinetic injection enhancement for environmental samples, a prior cleanup of the sample using standard ion-exchange cartridges is necessary. This cleanup step uses sequential cartridges to remove sulfate (barium cartridge), chloride (silver cartridge), and cations (H+ cartridge). Using this approach, detection limits for these four acids were as low as 1-2 micrograms/L for water samples and 25-50 micrograms/L for aqueous leachates of soil samples (10 mL of leachate/1.5 g of soil). The utility of this method for separation of CWA degradation products by CE is discussed in terms of pressure injection versus electokinetic injection. The effects of voltage and time of injection on the separation were investigated. Results from three types of soils and four types of water (groundwater, artificial seawater, tap water, bay water) indicated that the method has potential for environmental monitoring. Quantitative CE analysis with electrokinetic injection enhancement of detection limits of these types of environmental samples requires the use of an appropriate internal standard approach. The data presented here indicate that an internal standard-based approach could be expected to give analysis results in the sub-part-per-million concentration range of 90-110% of the true value.

Liquid Chromatographic Determination of Major Secondary Metabolites Produced by Aspergillus Species from Section Flavi

Abstract A liquid chromatographic (LC) method was developed for simultaneous determination of major secondary metabolites—including cyclopiazonic acid (CPA), O-methylsterigmatocystin (OMST), and the versicolorins—produced by Aspergillus species from section Flavi (A. flavus, A. parasiticus, A. tamarii, and A. caelatus) on a liquid medium. Metabolites were extracted with chloroform and quantitated without prior cleanup by means of normal- phase ion-pair partition LC on silica gel with a mobile phase of n-heptane-2-propanol-n-butanolwater- tetrabutylammonium hydroxide (2560 + 900 + 230 + 32 + 8, v/v). Recoveries of CPA and OMST from fungal cultures spiked at 10 μg/mL were 98.90 + 3.27 and 95.92 + 5.27% (n = 5), respectively. At spike levels of 100 μg/mL, recoveries were 98.89 + 3.87 and 97.65 + 4.32% (n = 5), respectively. Limits of detection for pure standards were 0.25 μg/mL for CPA (at 280 nm) and 0.30 μg/mL for OMST (at 310 nm). UV detector responses to CPA and OMST were linear to about 0.5 and 3.5 μg/injection, respectively

Determination of amino acids in biological fluids by capillary gas chromatography with nitrogen-phosphorus selective detection

A selective and sensitive method for the determination of protein and non-protein amino acids in biological fluids by capillary gas chromatography (GC) has been developed. The amino acids in the samples were directly converted into their N( O, S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with nitrogen-phosphorus selective detection (NPD) using a DB-17ht capillary column. Using this method, the derivatives of the 21 protein amino acids and the 25 non-protein amino acids provided excellent NPD responses and were quantitatively and reproducibly resolved within 28 min. The lower detection limits of these amino acids, at a signal-to-noise ratio of 3, were ca. 6–150 pg injected. The calibration curves for each amino acid in the range of 0.02–2 μg were linear and sufficiently reproducible for quantitative analysis. This method was successfully applied to small urine and serum samples without prior clean-up; there was no evidence of interference from coexisting substances. Overall recoveries of amino acids added to urine and serum samples were 83–112%. The intra-assay and inter-assay R.S.D. of amino acids in these samples were 0.3–8.9% ( n = 3) and 1.9–15.8% ( n = 3) respectively.