Quantitative Analysis Of Organic Acids Research Articles

Development and validation of a UHPLC-ESI-MS/MS method for the simultaneous determination of organic acids and phenolic compounds in Filipendula vulgaris, Polygonum divaricatum, Hypericum linarioides, and Rheum ribes

This research focused on investigating biologically active and pharmaceutically important phytochemicals using reliable and reproducible analytical techniques specifically designed for traditional medicinal plants. Considering the complex chemical diversity present in plant extracts, reliable identification methods are crucial. The goal of this study was to develop and validate a robust UHPLC-ESI-MS/MS method for the qualitative and quantitative analysis of organic acids (OAs) and phenolic compounds (PCs). With gradient elution of water containing 0.1 % formic acid and acetonitrile containing 0.1 % formic acid as the mobile phase, OAs and PCs were separated chromatographically on a C18 reversed-phase column. Through the use of an electrospray ionization source operating in negative ionization mode, these compounds were identified on a triple quadrupole tandem mass spectrometer. The validation results clearly indicate that the method exhibited linearity with a squared correlation coefficient (R2 ≥ 0.9986), demonstrating sensitivity (LOQ: 1.08 µg/L-10.79 µg/L), precision (RSD%≤2.73), and trueness (RE% ≤ 1.53) for the OAs and PCs found in the plants. The developed UHPLC-ESI-MS/MS method was successfully applied to the qualitative and quantitative analysis of OAs and PCs in Turkish plants, which are commonly used in traditional medicine. Our study identified a total of 12 OAs and PCs in F. vulgaris, 12 in P. divaricatum, 9 in H. linarioides, and 11 in R. ribes. In particular, gallic acid had the highest content in F. vulgaris (925.67 µg/g extract) and R. ribes (1358.78 µg/g extract), while H. linarioides (730.66 µg/g extract) and P. divaricatum (4003.02 µg/g extract) had relatively high levels of quinic acid. In conclusion, the developed UHPLC-ESI-MS/MS method allows the simultaneous analysis and quantification of 2 OAs and 33 PCs in plants and proves to be a valuable tool for evaluating the efficacy of traditional medicinal plants.

Fluorescent sensor array constructed by functionalized carbon nanodots for qualitative and quantitative analysis of urinary organic acids biomarkers

Adequate assessment of the level of organic acids (OA) in human urine is significant for non-invasive diagnosis and monitoring of OA-related diseases. However, the common lock-and-key sampling method for specific OA analysis is dramatically perturbed by the complicated chemical components in urine. A comprehensive qualitative and quantitative analysis of OAs in urine samples remains a significant challenge. Herein, a cross-reactive fluorescent sensor array based on amino acid-functionalized carbon nanodots (CDs) was designed for simultaneous qualitative and quantitative analysis of seven kinds of OAs, including homovanillic acid (HVA), vanillylmandelic acid (VMA), 3-hydroxyisovaleric acid (3-HA), lactic acid (LA), pyruvic acid (PA), glutaric acid (GA), and methylmalonic acid (MMA). The array system can specifically recognize multiple OAs in urine samples over a wide concentrations range (0.1–1000 μM) with 100% accuracy. The wide concentration range and high accuracy of the CD array system for OA analysis depend on a distinct fluorescence response from the differential binding affinity of various OAs to the CDs. The excellent recognition ability and selectivity of the sensing system were further confirmed by the double-blind and interference experiments. The sensor array achieved satisfactory distinguishing effects of separate OAs and binary OA samples (HVA/VMA) in urine samples. In particular, the quantitative analysis of the one-OA system or two-OA system (HVA/VMA) can be realized by the array system. These results confirm that the sensor array based on the CDs is an efficient platform for OA analysis and a convenient tool for the non-invasive diagnosis of OA-related diseases.

Quantitative analysis of organic acids in pomelo fruit using FT-NIR spectroscopy coupled with network kernel PLS regression

Pomelo is a high-class characteristic food in China. The fruit quality of pomelo is much important for people’s health and sense of taste. After being picked, the fruit quality deteriorates because of the spontaneous physiological reactions due to the metabolism and changes of organic acids (OA). The detection of OA concentration is essential for evaluating the pomelo fruit quality. In this paper, the OA concentrations in pomelo fruit samples were quantitatively determined by using Fourier transform near-infrared (FT-NIR) spectroscopy coupled with the kernel partial least square (kernel PLS) method. The network architectural kernel was proposed for extracting the spectral feature data in the machine learning manner. The number of network hidden nodes were tuned in combination of the optimally selection of the PLS latent variables. An error-feedback iteration mechanism was used for self-adaptively training the network linking weights. Model calibration results show that the most suitable network structure was identified with 130 hidden nodes and 20 output nodes. The optimal kernel improved the PLS model with 8 feature latent variables. The model training results were acquired with the root mean square error as 0.834 g/kg, and the correlation coefficient as 0.936. In comparison with common kernel functions, the network kernel performed better in the model training part and in the testing part. The model prediction results indicated that the proposed automatically training of the network architectural kernel is feasible to improve the PLS modeling effects for the FT-NIR rapid analysis of OA in pomelo fruit.

Investigation of Organic Acids in Saffron Stigmas (Crocus sativus L.) Extract by Derivatization Method and Determination by GC/MS

The beneficial health properties of organic acids make them target compounds in multiple studies. This is the reason why developing a simple and sensitive determination and investigation method of organic acids is a priority. In this study, an effective method has been established for the determination of organic (lactic, glycolic, and malic) acids in saffron stigmas. N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was used as a derivatization reagent in gas chromatography combined with mass spectrometric detection (GC/MS). The saffron stigmas extract was evaporated to dryness with a stream of nitrogen gas. The derivatization procedure: 0.1 g of dried extract was diluted into 0.1 mL of tetrahydrofuran, then 0.1 mL MTBSTFA was orderly and successively added into a vial. Two different techniques were used to obtain the highest amount of organic acid derivatives from saffron stigmas. To the best of our knowledge, this is the first report of the quantitative and qualitative GC/MS detection of organic acids in saffron stigmas using MTBSTFA reagent, also comparing different derivatization conditions, such as time, temperature and the effect of reagent amount on derivatization process. The identification of these derivatives was performed via GC-electron impact ionization mass spectrometry in positive-ion detection mode. Under optimal conditions, excellent linearity for all organic acids was obtained with determination coefficients of R2 > 0.9955. The detection limits (LODs) and quantitation limits (LOQs) ranged from 0.317 to 0.410 µg/mL and 0.085 to 1.53 µg/mL, respectively. The results showed that the highest yield of organic acids was conducted by using 0.1 mL of MTBSTFA and derivatization method with a conventional heating process at 130 °C for 90 min. This method has been successfully applied to the quantitative analysis of organic acids in saffron stigmas.

Fast and accurate quantitative organic acid analysis with LC-QTOF/MS facilitates screening of patients for inborn errors of metabolism

Since organic acid analysis in urine with gaschromatography-mass spectrometry (GC-MS) is a time-consuming technique, we developed a new liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) method to replace the classical analysis for diagnosis of inborn errors of metabolism (IEM). Sample preparation is simple and experimental time short. Targeted mass extraction and automatic calculation of z-scores generated profiles characteristic for the IEMs in our panel consisting of 71 biomarkers for defects in amino acids, neurotransmitters, fatty acids, purine, and pyrimidine metabolism as well as other disorders. In addition, four medication-related metabolites were included in the panel. The method was validated to meet Dutch NEN-EN-ISO 15189 standards. Cross validation of 24 organic acids from 28 urine samples of the ERNDIM scheme showed superiority of the UPLC-QTOF/MS method over the GC-MS method. We applied our method to 99 patient urine samples with 32 different IEMs, and 88 control samples. All IEMs were unambiguously established/diagnosed using this new QTOF method by evaluation of the panel of 71 biomarkers. In conclusion, we present a LC-QTOF/MS method for fast and accurate quantitative organic acid analysis which facilitates screening of patients for IEMs. Extension of the panel of metabolites is easy which makes this application a promising technique in metabolic diagnostics/laboratories.

Development of a quantitative method for organic acid in wine and beer using high performance liquid chromatography.

This study was conducted to compare the organic acid content in liquors (red wine, white wine, and beer) using three different high-performance liquid chromatography analysis methods. Post-column reaction methods (method 2 and 3) were found to be more promising than UV-detection method (method 1). Using method 2 (two columns), the analyzed red wine was found to contain 2,652.4 mg/L tartaric acid and 1,392.9 mg/L lactic acid but relatively lower amounts of malic acid (271.0 mg/L). Furthermore, tartaric acid (1,160.8-2,749.1 mg/L) and malic acid (470.2-3,107.9 mg/L) were the major components in white wine. Beers were analyzed using method 3 (one column). In the analyzed foreign beers, lactic acid (95.9-226.4 mg/L), malic acid (62.2-110.5 mg/L), acetic acid (93.5-183.8 mg/L), and succinic acid (37.0-56.2 mg/L) were detected. Similar to the foreign beers, the contents of succinic acid in the domestic beers were the lowest. The proposed methods could be useful for quantitative analysis of organic acids in wine and beer.

LC with Evaporative Light-Scattering Detection for Quantitative Analysis of Organic Acids in Juices

In this work, a performing LC method with evaporative light-scattering detector was described for the analysis of tartaric, malic, ascorbic, citric and succinic acids in fruit juices. The method was optimized and validated in comparison with LC-UV, in terms of accuracy, LODs, LOQs and precision. The successive application to ACE, orange, pear, peach, mulberry and apple juices allowed separation and quantitative determination of organic acids in about 20 min just diluting and filtering the sample before LC determination. In the analysed juices, citric, malic and ascorbic acids were always present, and in all the samples, citric acid was the prevailing acid followed by malic acid except for peach and apple juices where an opposite trend was found. Tartaric and succinic acids were detected at lower concentrations and more rarely, except for peach juices, all containing tartaric acid, and ACE and orange juices, all containing succinic acid.

THE USE OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AS SCREENING TECHNIQUE FOR PECTIN AND PECTIN SUBSTANCES OF DIETARY FIBERS

The peak of the unspecified compound of quantitative analysis of organic acids in biological materials by using high-performance liquid chromatography was identified. This peak corresponded to polygalacturonic acid. A High-performance liquid chromatographic method was developed to identify and quantify of water soluble pectin fraction and insoluble pectin fraction, galacturonic acid and polygalacturonic acid. The method based on the extraction of free water soluble fraction with phosphate buffer (pH 4.8) or with 0.01 N phosphoric acid and on the acid hydrolysis of insoluble pectin fraction in presence 0.1 N HNO3. The pectin fractions were separated and determined on a C610H column using elution with 0.01 N phosphoric acids and ultraviolet detection at 210 nm. Galacturonic acid and polygalacturonic acid were used as internal standard. The calibration curves were presented for each of the standards. The determinations were performed in the linear range of 0.04÷0.17% w/v for both pectin fractions. Proposed procedure can considered as a rapid method for quantitative pectin determination in the food, food nutrition’s food, and pharmaceutical agents.

Direct quantification of organic acids in aerosols by desorption electrospray ionization mass spectrometry

Desorption electrospray ionization mass spectrometry (DESI-MS) is a novel analytical technique for direct surface analysis under ambient conditions. In this work, we report the first application of DESI-MS to the analysis of organic acids in atmospheric aerosols. We took oxalic and oleic acids as two typical representatives of the particulate organic acids. Experimental parameters for DESI-MS were optimized to enhance the signal intensity. Calibrations for both acids showed great linearity over a concentration range of 5 orders of magnitude with correlation coefficients R 2>0.99. The relative standard deviation (RSD) was less than 10% for oxalic acid and within 15% for oleic acid measurement. The detection limits for oxalic acid and oleic acid are about 1 pg mm −2 with 5–10 s sampling time. Mass concentrations of organic acids in both laboratory-generated biomass burning and ambient aerosols were measured by DESI-MS and the results agree well with the ion chromatography (IC) and GC–MS values. This work demonstrates that DESI-MS is a promising method for rapid quantitative analysis of organic acids in atmospheric aerosols.

Development of a capillary electrophoresis‐mass spectrometry method using polymer capillaries for metabolomic analysis of yeast

Metabolomics is an emerging field in analytical biochemistry, and the development of such a method for comprehensive and quantitative analysis of organic acids, carbohydrates, and nucleotides is a necessity in the era of functional genomics. When a concentrated yeast extract was analyzed by CE-MS using a successive multiple ionic-polymer layer (SMIL)-coated capillary, the adsorption of the contaminants on the capillary wall caused severe problems such as no elution, band-broadening, and asymmetric peaks. Therefore, an analytical method for the analysis of anionic metabolites in yeast was developed by pressure-assisted CE using an inert polymer capillary made from poly(ether etherketone) (PEEK) and PTFE. We preferred to use the PEEK over the PTFE capillary in CE-MS due to the easy-to-use PEEK capillary and its high durability. The separation of anionic metabolites was successfully achieved with ammonium hydrogencarbonate/formate buffer (pH 6.0) as the electrolyte solution. The use of 2-propanol washing after every electrophoresis run not only eliminated wall-adsorption phenomena, but allowed for good repeatability to be obtained for migration times in the metabolomic analysis.

A Pyruvate Cycling Pathway Involving Cytosolic NADP-dependent Isocitrate Dehydrogenase Regulates Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) from pancreatic islet beta-cells is central to control of mammalian fuel homeostasis. Glucose metabolism mediates GSIS in part via ATP-regulated K+ (KATP) channels, but multiple lines of evidence suggest participation of other signals. Here we investigated the role of cytosolic NADP-dependent isocitrate dehydrogenase (ICDc) in control of GSIS in beta-cells. Delivery of small interfering RNAs specific for ICDc caused impairment of GSIS in two independent robustly glucose-responsive rat insulinoma (INS-1-derived) cell lines and in primary rat islets. Suppression of ICDc also attenuated the glucose-induced increments in pyruvate cycling activity and in NADPH levels, a predicted by-product of pyruvate cycling pathways, as well as the total cellular NADP(H) content. Metabolic profiling of eight organic acids in cell extracts revealed that suppression of ICDc caused increases in lactate production in both INS-1-derived cell lines and primary islets, consistent with the attenuation of pyruvate cycling, with no significant changes in other intermediates. Based on these studies, we propose that a pyruvate cycling pathway involving ICDc plays an important role in control of GSIS.

Reye syndrome after acute enteritis during the neonatal period

Purpose:The prevalence of Reye syndrome has decreased since late 1980's. But we report that recently there were concentrative attacks of Reye syndrome after acute enteritis during the neonatal period. Methods:Clinical symptoms and laboratory results(quantitative organic acid analysis, routine chemistry, arterial blood gas analysis, serum ammonia) of seven patients admitted at the Samsung Medical Center, Sanggye Paik Hospital, Wonju Christian Hospital and Chuncheon Sacred Heart Hospital, referred from Jan. 2005 to Apr. 2005, were analysed retrospectively. The major clinical symptoms were derived from the patients' clinical records sended with urine samples and quantification of organic acids were done with gas chromatography and mass spectrometry. Results:The mean age of seven cases is 18 days and the major preceding symptoms were gastrointestinal symptoms(vomiting, diarrhea, refusal to feeding). The major clinical symptoms were clouded conciousness, repiratory difficulty, vomiting, seizures, and diarrhea. One patient died; that patient's serum ammonia was twenty times higher than normal. Conclusion:The seven patients were neonates. Reye syndrome has been known to be closely related with upper respiratory infections as a preceding disease and to internal use of aspirin, but in our study, the major preceding disease of the seven cases was gastrointestinal infection and none of these used aspirin.

Organic acidemias in Korea - Eight years experience of organic acid analysis -

Purpose:We have done this retrospective study to know the relative incidence and clinical manifestations of organic acidopathies in Korea during 8 years(from Jul. 1997 to May 2005). This results of organic acid analysis of 1,787 patients were compared with the results of organic acid analysis that were published three years ago. Methods:The results of quantitative organic acid analysis of samples of 1788 patients, referred from Jul. 1997 to May 2005, were analyzed retrospectively according to four age group(-2 mon, 3 mon-2 years, 3-12 years) and major clinical manifestations. Quantification of 83 organic acids was done with gas chromatography and mass spectometry. Results:We diagnosed 470 patients with 27 diseases of organic acid metabolism during this study period. Diseases found more than 10 cases are cytosolic 3-ketothiolase deficiency, mitochondrial respiratory chain disorders, PDHC deficiency, mitochondrial 3-ketothiolase deficiency, glutaric aciduria type II, biotinidase deficiency, methylmalonic aciduria and propionic aciduria. Other diseases were diagnosed in less than 10 cases. Conclusion:Though the incidence of individual organic acidemia is low, the overall incidence of organic acidemia as a whole seems to be relatively high in Korea. Compared with the results of organic acid analysis that were reported three years ago, we couldn't find a new disease and the difference of the relative incidences of high incident diseases. We were apprehensive of the errors that was owing to the short study period(3 years), but the relative incidences of our study(8 years) were similar to the results of organic acid analysis that were reported three years ago.

Metabolomic approaches to mitochondrial disease: correlation of urine organic acids

In order to examine correlations which might be useful in ascertaining or confirming the diagnosis of mitochondrial disease, a retrospective analysis of urine organic acids was performed. Among 3646 analyses from randomly selected samples referred to our laboratory, there were 258 specimens from 67 patients with various known disorders of mitochondrial oxidative function, most of whom were known to have chronic and persistent elevations of blood lactic acid, and 176 samples from 21 patients with diagnosed organic acidemia. Urine lactate was not a useful discriminator; only 7.6% of results from infants with mitochondrial disease fell the 95th percentile for patients without mitochondrial disease. Most of the Krebs cycle intermediates were also not useful in discriminating patients with mitochondrial disorders. Interestingly, there was strikingly poor correlation among most of those analytes in all patient groups, but fumarate and malate were uniquely well correlated (r 2=0.840). Fumarate and malate were also the most useful in distinguishing patients with mitochondrial disease and organic acidemia from the pool of unselected or undiagnosed patients, although the utility was somewhat limited. Using a cutoff value of approximately 90 mmol/mol creatinine for fumarate or malate at age <1 year, or a cutoff of approximately 25 for older patients, 25ndash;30% of mitochondrial disease patients can be distinguished with a 5% false positive rate. Further refinements to this approach may better characterize the metabolomic profile and may improve the diagnostic utility of quantitative organic acid analysis in mitochondrial disease.

Physiology and pathophysiology of organic acids in cerebrospinal fluid.

Concentrations of organic acids in cerebrospinal fluid (CSF) appear to be directly dependent upon their rate of production in the brain. There is evidence that the net release of short-chain monocarboxylic acids from the brain is a major route for removing these products of cerebral metabolism. Concentrations of organic acids in blood and CSF are largely independent of each other. Quantitative reference values for the concentrations of organic acids in CSF and plasma as well as ratios of individual organic acids between CSF and plasma were determined in 35 pairs of samples from paediatric patients. Over 25 organic acids were quantifiable in all or in the majority of CSF and/or plasma specimens (limit of detection 1 mumol/L). There were substantial differences in the CSF/plasma ratios between subgroups of organic acids. Metabolites related to fatty-acid oxidation were present in CSF in substantially less amounts than in plasma. Organic acids related to carbohydrate and energy metabolism and to amino acid degradation were present in CSF in the same amounts as or slightly smaller amounts than in plasma. Finally, some organic acids were found in substantially higher amounts in CSF than in plasma, e.g. glycolate, glycerate, 2,4-dihydroxybutyrate, citrate and isocitrate. Studies of organic acids in CSF and plasma samples are presented from patients with 'cerebral' lactic acidosis, disorders of propionate and methylmalonate metabolism, glutaryl-CoA dehydrogenase deficiency and L-2-hydroxy-glutaric aciduria. It became apparent that derangements of organic acids in the CSF may occur independently of the systemic metabolism. Quantitative organic acid analysis in CSF will yield new information on the pathophysiology in the central nervous system (CNS) of these disorders and may prove necessary for successful monitoring of treatment of organoacidopathies, which present mainly with neurological disease. For example, in glutaryl-CoA dehydrogenase deficiency the urinary excretion of glutarate appears to be an inadequate parameter for monitoring the effect of dietary therapy, without plasma and CSF determinations. In L-2-hydroxyglutaric aciduria the elevation of L-2-hydroxyglutarate was found to be greater in CSF than in plasma. In addition, some other organic acids, glycolate, glycerate, 2,4-dihydroxybutyrate, citrate and isocitrate, were also elevated in the CSF of the patients out of proportion to normal levels in plasma and urine. High concentrations of an unknown compound, which was tentatively identified as 2,4-dihydroxyglutarate, were found in the CSF of patients with L-2-hydroxyglutaric aciduria.(ABSTRACT TRUNCATED AT 400 WORDS)

STUDIES ON YEAST DIFFERENTIATION USING ORGANIC ACID METABOLITES PART 1. DEVELOPMENT OF METHODOLOGY USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

The separation of organic acids on the column Aminex HPX-87H from Bio-Rad is described. The relationship between column temperature and retention time was studied for both sulphuric and phosphoric acid eluants and an analytical scheme using 0.2% (v/v) phosphoric acid was developed. While the procedure is designed for the commonly occurring non-volatile acid metabolites it was demonstrated that conditions could be established for the analysis of fatty acids to C10. Difficulties in the qualitative and quantitative analysis of organic acids in beer are discussed.

Determination of Organic Acids in Dairy Products by High Performance Liquid Chromatography

ABSTRACTA high performance liquid chromatographic method was developed for the quantitative analysis of organic acids in dairy products. A reverse‐phase C8 column at room temperature, a mobile phase of 0.5% w/v buffer ((NH4)2HPO4 at pH 2.24 with H3, PO4)‐0.4% v/v acetonitrile, UV detection at 214 nm and 1.2 mL/min flow rate were utilized. Formic, acetic, pyruvic, propionic, uric, orotic, citric, lactic and butyric acids were quantitated for raw milk, yogurt, Blue, Provolone, Port Salut and Quartirolo cheeses. Recoveries greater than 85.3% were observed for all acids.

Characterization of biotechnological processes and products using high‐performance liquid chromatography (HPLC). III. Quantitative analysis of organic acids in the α‐ketoglutaric acid synthesis

AbstractA HPLC technique for the analysis of organic acids in the production of α‐ketoglutaric acid was developed. The method was applied and optimized for the quantitative determination of citric acid, pyruvic acid, isocitric acid and α‐ketoglutaric acid in fermentation solutions.As microorganism the yeast Yarrowia lipolytica and as substrates glucose or paraffins were used.The chromatographic separations were carried out by means of 50 and 100 × 8 mm i.d. glass columns packed with an anion‐exchange resin based on an 8% cross‐linked polystyrene‐divinylbenzene copolymer.The relative errors ranged from 2.1% (α‐ketoglutaric acid) to 5.2% (isocitric acid). The percent recovery values varied between 94.4% (isocitric acid) and 107.7% (pyruvic acid).The contents of organic acids in fermentation solutions after the microbial synthesis based on paraffins or glucose were compared.

Determination of organic acids in sugar cane process juice by high-performance liquid chromatography: improved resolution using dual aminex HPX-87H cation-exchange columns equilibrated to different temperatures

The application of high-performance liquid chromatography to the quantitative analysis of organic acids in sugar cane process juice is described. Separation of these acids is achieved on Aminex HPX-87H cation-exchange columns. Resolution is improved by connecting two columns in series and equilibrating them at different temperatures. The acids in the sample are first isolated on DEAE-Sephadex A-25 anion-exchange resin equilibrated with 0.5 M isobutyric acid. They are then eluted with 0.5 M sulphuric acid and injected directly onto the HPLC column after filtration through a 0.45-μm membrane. This method allows the simultaneous determination of oxalic, cis-aconitric, citric, phosphoric, malic, trans-aconitic, succinic, glycolic, lactic, formic and acetic acids. Isobutyric acid is also separated on this system and provides a useful guide to the loading capacity of the anion-exchange resin.

High Performance Liquid Chromatographic Determination of Organic Acids in Dairy Products

ABSTRACTA simple isocratic HPLC technique was developed for the quantitative analysis of organic acids in dairy products. An Ammex HPX‐ 87 column at 65°C, 0.0090N H2SO4 mobile phase and UV detection at 220 and 275 nm were utilized. Orotic, citric, pyruvic, lactic, uric, formic, acetic, propionic, butyric, and hippuric acids were quantitated for whole milk, skim powder, cultured buttermilk, sour cream, cottage cheese, yogurt, sharp Cheddar cheese, and blue cheese. Over 90% recoveries of acids added to whole milk were observed for ail acids except butyrid; the average recovery for butyric was 86%.