High-performance Liquid Chromatrography Research Articles

An efficient high-speed countercurrent chromatography method for preparative separation of javanicin from Fusarium solani, a fungus isolated from the fruiting body of the mushroom Trametes trogii.

To develop an efficient method for large preparation of javanicin from Fusarium solani, a rapid and simple method by high-speed countercurrent chromatography was established based on average polarity (P' values) and partition coefficients (K values) of crude samples. A suitable solvent system for high-speed countercurrent chromatography was selected from many possible biphasic solvent systems. HSCCC was successfully applied to separate and purify javanicin, the main bioactive component of solid cultures of the fungus F. solani isolated from the fruiting body of Trametes trogii, with petroleum ether-ethyl acetate-methanol-water (4:3:2:1, v/v) as solvent system. A total amount of 40.6 mg of javanicin was obtained from 100 mg crude sample. The purity of javanicin was 92.2% with a recovery of 95.1%, as determined by high-performance liquid chromatrography. The molecular structure was identified primarily by NMR and MS methods. The results indicated that high-speed countercurrent chromatography could be a powerful technology for separating naphthoquinones from the solid cultures of the fungus F. solani. It is also of significance that the separation of javanicin from natural source was carried out for the first time utilizing high-speed countercurrent chromatography.

Removal of bias in C37 alkenone-based sea surface temperature measurements by high-performance liquid chromatography fractionation

Sea surface temperature determinations based on marine sedimentary C37 alkenone distributions have provided a wealth of data for paleoclimatic studies, including those performed at high resolution. The success of this approach results from several characteristics of alkenone compounds, e.g. their geochemical properties (such as unequivocal synthesis by certain widespread haptophyte algae, plus chemical stability/preservation of the original alkenone distributions during sedimentation), and their analytical properties (such as fast clean up procedures using alkaline hydrolysis of sediment extracts, followed by robust instrumental methods allowing large scale sample processing). Here we show that, in sediments under the influence of continental inputs, coelution of these compounds with cholest-5-enyl 3β-undecenyl ether and 24-methylcholesta-5,22-dienyl 3β-undecenyl ether deviate the SST measurements despite alkaline hydrolysis. Here, we report a new high performance liquid chromatrography fractionation method which eliminates these interfering compounds and gathers all the alkenones into a single fraction. These fractions can then be analysed by gas chromatography as in the initial approach, providing large amounts of data as required in high resolution studies.

Inhibitory Potential of Cocoa Leaves Polyphenolics-Rich Extract on Xanthine Oxidase and Angiotensin 1-Converting Enzyme

The over-activities of xanthine oxidase (XO) and angiotensin 1-converting enzyme (ACE) contribute to the pathogenesis of gout and hypertension, respectively. Polyphenolics and other natural inhibitors of XO and ACE have been shown to be useful for managing these two diseases. Therefore, this study evaluated the inhibitory potential of cocoa leaves polyphenolics-rich extract on XO and ACE, and its free radicals (2,2- diphenylpicrylhydrazyl [DPPH*] and 2,2´-azino-bis-3-ethylbenzthiazoline-6-sulphonic [ABTS*+]) scavenging ability, in vitro. The XO and ACE inhibition; and DPPH* and ABTS*+ scavenging assays were carried out using Spectrophotometric methods. Flavonoids and phenolic acids content of the extract was quantified using high performance liquid chromatrography coupled with diode array detection (HPLC-DAD). The extract strongly inhibited XO and ACE in a dose dependent pattern, with half-maximal inhibitory concentration (IC50) of 33.15 ± 1.84 μg/mL and 18.72 ± 1.05 μg/mL, respectively. The extract also scavenged DPPH* and ABTS*+. The HPLC-DAD analysis of the extract revealed the presence of flavonoids including catechin, rutin, quercetin and apigenin; and phenolic acids including gallic, caffeic and p-coumaric acids; with quercetin and caffeic acid as the most abundant flavonoid and phenolic acid , respectively. Cocoa leaves may be useful for preventing and managing gout and hypertension, through XO and ACE inhibition.

Structure and Property Changes of Transglutaminase-Induced Modification of Sodium Caseinate in the Presence of Oligochitosan of 5 kDa

Sodium caseinate was modified by transglutaminase-catalyzed reaction in the presence of oligochitosan of 5 kDa at two different levels, aiming to generate two glycated and crosslinked caseinate products (GC-caseinate I and II) and to clarify their respective structure and property changes. Electrophoretic analysis with aid of protein and saccharide staining confirmed that both GC-caseinate I and II were glycated and crosslinked protein products, while high-performance liquid chromatrography analysis demonstrated that both GC-caseinate I and II contained glucosamine (12.8 and 30.8 g/kg protein, respectively). In comparison with sodium caseinate, GC-caseinate I and II also contained less reactable –NH2 groups (0.50 and 0.52 versus 0.62 mol/kg protein), more –OH groups in their molecules, but they had more ordered secondary structure than sodium caseinate. In addition, GC-caseinate I and II showed higher water-binding capacity, larger hydrodynamic radius (173.9 and 168.2 versus 145.3 nm), and larger negative zeta-potential (–32.9 and –30.9 versus –27.6 mV) in aqueous dispersions, but they exhibited lower thermal stability (namely, greater mass loss) at temperature higher than 286ºC. Oligochitosan-glycated sodium caseinate at lower and higher extents was observed to be unfavorable and favorable to the in vitro digestibility of GC-caseinate I and II, respectively. It is concluded that application of transglutaminase and the oligochitosan can modify structure and property of sodium caseinate to generate new protein ingredients with good hydration and colloidal stability.

Phenolics extract of Tetrapleura tetraptera fruit inhibits xanthine oxidase and Fe2+-induced lipid peroxidation in the kidney, liver, and lungs tissues of rats in vitro

The phenolics composition and inhibitory activity of Tetrapleura tetraptera fruit extract on xanthine oxidase (XO) and Fe2+-induced lipid peroxidation in the kidney, liver and lungs tissues of rats were evaluated in vitro. Phenolics (flavonoids and phenolic acids) were quantified using reverse-phase high performance liquid chromatrography coupled with diode array detection (HPLC-DAD). The XO and Fe2+-induced lipid peroxidation inhibitory abilities of the extract were evaluated using spectrophotometric methods. The extract contained some flavonoids and phenolic acids, including catechin, epicatechin, rutin, quercetin, luteolin, apigenin; gallic, chlorogenic, caffeic and ellagic acids that are beneficial to health. The extract inhibited XO in the kidney, liver and lungs tissues in a dose-dependent manner. The half-maximal inhibitory concentrations (IC50) of the extract on XO from the tissues varied significantly (P<0.05), and were in the order of liver>kidney>lungs. The extract also inhibited Fe2+-induced lipid peroxidation in a dose-dependent pattern, having IC50 in the order of liver>lungs>kidney. T. tetraptera fruit extract could be a promising nutraceutical for preventing and managing hyperuricaemia and the associated disease conditions, due to its ability to inhibit XO; this bioactivity is attributable to combined effect of its flavonoids and phenolic acids.

Validated HPLC-Fl Method for the Analysis of S-Adenosylmethionine and S-Adenosylhomocysteine Biomarkers in Human Blood

The natural methyl donor group, S-adenosylmethionine and its product, S-adenosylhomocysteine play an important role in many biochemical reactions involving transmethylation reactions. These compounds can be used as biomarkers in incipient diagnosis of various pathological disorders therefore the validation of a suitable method to routinely analysis of these compounds is very important. In this paper, a high performance liquid chromatrography method for S-adenosylmethionine and S-adenosylhomocysteine measurement as fluorescent 1,N(6)-ethanoderivatives from biological samples was validated in terms of selectivity, linearity range of the response (R > 0.9993), detection limit (9 × 10(-9) and 4.4 × 10(-9) mol L(-1)), the limit of quantitation (9.7 × 10(-9) and 5.7 × 10(-9) mol L(-1)), precision, trueness and robustness. The method for quantification simultaneous of these compounds is rapid, sensitive and precise and appropriate for clinical analysis.

Influence of acetaminophen consumption and exercise on Achilles tendon structural properties in male Wistar rats

Chronic consumption of acetaminophen (APAP) during exercise training leads to a reduction in tendon stiffness and modulus compared with a placebo. We explored whether this effect could be due to a reduction in tendon collagen content or cross-linking. Ten-week-old male Wistar rats (n = 50) were divided into placebo or APAP groups and into sedentary or treadmill-exercised groups. APAP (200 mg/kg) or saline was administered once daily by oral gavage. Rats in the exercise groups ran on a treadmill 5 days per week for 8 wk with progression to 60 min per day, 20 m/min, and 8° incline. After 8 wk, lyophilized Achilles tendon samples were assayed for the collagen-specific amino acid hydroxyproline and cross-linking [hydroxylyslpyridinoline (HP)] content by high-performance liquid chromatrography. Collagen content was not influenced by exercise or APAP (P > 0.05). Compared with placebo, tendon water content was 7% (P = 0.006, main effect) lower in animals consuming APAP (placebo: 54.79 ± 0.8%, APAP: 50.89 ± 1.2%). HP in the Achilles tendon was 36% greater (sedentary: 141 ± 15, exercise: 204 ± 26 mmol/mol collagen) in the exercise-trained rats independent of drug treatment (P = 0.020, main effect). Independent of exercise, HP content was 33% lower (P = 0.032, main effect) in the animals consuming APAP (placebo: 195 ± 21, APAP: 140 ± 19 mmol/mol collagen). Our data suggests that chronic consumption of APAP results in a reduction in collagen cross-linking and a loss of tissue water independent of chronic exercise. This reduction in cross-linking and water content could contribute to the decrease in tendon stiffness noted in humans chronically consuming APAP.

Bioassay of Safinamide in Biological Fluids of Humans and Various Animal Species

This paper describes three methods to bioassay safinamide (CAS 133865-89-1) in biological fluids of humans and laboratory animals for pharmacokinetic, toxicokinetic and bioavailability studies. Two methods profited from liquid chromatography tandem mass spectrometry (LC-MS-MS) system, one (micro bioassay) working in the low dynamic range 0.5-20 ng/ml, the other in the range 20-6000 ng/ml. A third method used high-performance liquid chromatrography with fluorimetric detection (HPLC-FD), working in the dynamic range 20-1000 ng/ml. All the three methods were validated in compliance with the accreditated views on analytical bioassays. The shorter run time (5.5 min vs 16 min) has led the authors to prefer the two LC-MS-MS methods to the HPLC-FD bioassay, even if all the performances of the three methods were excellent. The methods described in this paper were and are still now extensively used to assay safinamide in more than 10,000 specimens of biological fluids from humans and laboratory animals in the development program of the drug. Main pharmacokinetic results obtained in various Phase I trials on healthy volunteers are briefly reported.

The influence of acetaminophen and exercise on rat Achilles tendon structural properties

The purpose of this study was to evaluate the influence of acetaminophen (APAP) and exercise on the structural properties of rat Achilles tendon. Ten-week-old male Wistar rats were divided into placebo or APAP groups and further divided into sedentary or treadmill exercised groups. APAP (200 mg•kg−1) or placebo (saline) was administered once daily via oral gavage for 8 weeks. Tendon collagen content was determined by detection of the collagen specific amino acid hydroxyproline using high performance liquid chromatrography. Rats in the exercise group ran on a treadmill five days per week for 8 weeks with progression to 60 minutes per day, 20 m•min−1, and 8° incline. Exercise did not influence tendon collagen content and data were collapsed into placebo (n=24) and APAP (n=26) groups. Achilles tendon collagen content was greater (p<0.065) in the rats given APAP (placebo: 598±22; APAP: 652±18 μg collagen•mg tendon wet weight). When expressed relative to tendon dry mass collagen content was not influenced by APAP (placebo: 1346±60; APAP: 1345±57 μg collagen•mg tendon dry weight). Evaluation of tendon water content revealed a 7% (p<0.01) decrease in tendon water content in the APAP group when compared to placebo. Our data suggests that chronic consumption of APAP results in a loss of tissue water from tendon and this effect may be independent of exercise. Midwestern University College of Health Science and Office of Research and Sponsored Programs

Estudio de la determinación de la hemoglobina glucosilada en pacientes con diabetes mellitus 2 en sangre capilar en un centro de atención primaria

Objective To determine the reliability and practicability of the point-of-care- test (POCT) analyzer, Afinion, for capillary HbA1c testing. To assess the benefits of its implementation on the intra-annual follow up of type 2 diabetic patients. Design Descriptive cross-sectional study. Analytical validation of the Afinion reader. Setting Primary Health Care (CAP Carmel and Bon Pastor Clinic Laboratory). Participants A total of 94 type 2 diabetic patients selected according to their previous HbA1c value. Methods We performed one capillary puncture and one venous extraction on each visit. The capillary sample was assessed in real time on the Afinion in the Primary Health Care Centre and the venous sample was sent to Bon Pastor Clinic Laboratory for assessment on an Afinion analyzer and by a high performance liquid chromatrography (HPLC) reference method. Practicability was assesses by both by the operators of the Afinion and the patients using an 11 question questionnaire. The efficiency in terms of process timings was also evaluated. Results Intra-serial coefficient of variation (CV) was lower than 1% and inter-serial lower than 3%. The regression analysis showed: Afinion capillary sample = 0.95 Afinion venous + 0.21. No systematic or proportional error was detected in the 95% confidence interval (95% CI). The comparison between venous HPLC and Afinion showed: Afinion capillary sample = 0.80 HPLC + 1.14. A statistically significant difference was shown for these values at the 95% CI. Practicability was valued by users from 7 to 9.2 (professionals) and from 7.7 to 9.2 (patients). Implementation of the Afinion capillary method for intra-annual testing in follow up of diabetic patients could result in the saving of 600-900 professional hours/year. Conclusions Afinion seems to be a good choice for the intra-annual determination of HbA1c when compared to the traditional process due to its accessibility, practicability and efficiency. Professionals should know the limitations of the POCT method in order to consider the validity of the results.

The development of an activated carbon from cherry stones and its use in the removal of ochratoxin A from red wine

The ability of activated carbon (AC) prepared from cherry stones (CS) by activation with H 3PO 4, ZnCl 2 or KOH to remove ochratoxin A (OTA) from two Italian red wines has been studied. AC was characterized in terms of texture and surface chemistry. OTA was analyzed by reversed-phase high-performance liquid chromatrography, using a fluorescence detector. The content of OTA in the starting wines is 7.38 and 2.36 μg/L. The adsorption of OTA is high only for one AC, which was prepared by KOH activation at 900 °C, using the 3:1 KOH:CS impregnation ratio. It possesses a large apparent surface area ( S BET = 1620 m 2/g) and a high volume of large size macropores (1.84 cm 3/g). It also contains narrow mesopores and intermediate size and wide micropores. Its content of acidic oxygen surface groups is low, whereas the content of basic groups is high (2.62 meq/g). The treatment of the wines with such an AC results in a decrease of the initial OTA content of more than 50%. However, the changes produced in the total polyphenolic index, color intensity, and hue are small (i.e. ∼8%, ∼5.5% and ∼1.2%, respectively).

Biomonitoring of Perfluorinated Compounds in Children and Adults Exposed to Perfluorooctanoate-Contaminated Drinking Water

Objective40,000 residents in Arnsberg, Germany, had been exposed to drinking water contaminated with perfluorinated compounds (PFCs). Internal exposure of the residents of Arnsberg to six PFCs was assessed in comparison with reference areas.Design and participantsOne hundred seventy children (5–6 years of age), 317 mothers (23–49 years), and 204 men (18–69 years) took part in the cross-sectional study.MeasurementsIndividual consumption of drinking water and personal characteristics were assessed by questionnaire and interview. Perfluorooctanoate (PFOA), perfluorooctanesulfonate (PFOS), perfluorohexanoate, perfluorohexanesulfonate (PFHxS), perfluoropentanoate, and perfluorobutanesulfonate (PFBS) in blood plasma and PFOA/PFOS in drinking water samples were measured by solid-phase extraction, high-performance liquid chromatrography, and tandem mass spectrometry detection.ResultsOf the various PFCs, PFOA was the main compound found in drinking water (500–640 ng/L). PFOA levels in blood plasma of residents living in Arnsberg were 4.5–8.3 times higher than those for the reference population (arithmetic means Arnsberg/controls: children 24.6/5.2 μg/L, mothers 26.7/3.2 μg/L, men 28.5/6.4 μg/L). Consumption of tap water at home was a significant predictor of PFOA blood concentrations in Arnsberg. PFHxS concentrations were significantly increased in Arnsberg compared with controls (p < 0.05). PFBS was detected in 33% of the children, 4% of the women, and 13% of the men in Arnsberg compared with 5%, 0.7%, and 3%, respectively, in the reference areas (p < 0.05). Regression analysis showed that age and male sex were significant predictors of PFOS, PFOA, and PFHxS; associations of other regressors (diet, body mass index) varied among PFCs.ConclusionsPFC concentrations in blood plasma of children and adults exposed to PFC-contaminated drinking water were increased 4- to 8-fold compared with controls.

A chromatography method for the screening and confirmatory detection of dexamethasone

Corticosteroids, such as dexamethasone, have been illegally used as growth promoting agents to obtain an economical benefit from increased muscle development. These substances remain in meat and other animal products and may have negative toxic consequences for consumers. A screening and confirmatory method for dexamethasone detection in feed and drinking water in livestock has been developed and validated. This method is based on immunoaffinity chromatography followed by reverse-phase high-performance liquid chromatrography (IAC–HPLC) with diode array detection (DAD) at 242 nm. Flumethasone was used as internal standard. The method has been validated according to the European Union regulation 2002/657/EC for banned substances and, thus, the specificity, decision limit, detection capability, recovery and repeatability have been determined. The decision limits (CCα) were 26 ng/mL for water and 190 ng/g for feed while detection capabilities (CCβ) were 30 ng/mL for water and 217 ng/g for feed. The method showed good accuracy and precision. The stability of dexamethasone under frozen storage has been studied over a 3.5-month period. No losses were observed, thus confirming that water samples taken by inspectors can be stored frozen for a few weeks until analysis. This method has proved to be relatively simple and useful for rapid screening and confirmation of the presence of dexamethasone in water and feed used for meat-producing animals.

Covalent protein adduct formation and protein cross-linking resulting from the maillard reaction between cyclotene and a model food protein.

Covalent Maillard products of the reactions of carbonyl compounds with proteins are often described in the literature, but, until recently, evidence for their existence has been indirect. Cyclotene (2-hydroxy-3-methylcyclopent-2-enone), a common flavor compound, was incubated with a model food protein, ribonuclease, and found to cross-link the protein. Size exclusion high-performance liquid chromatrography and electrospray mass spectrometry of the early stages of the reaction provide strong evidence for covalent adducts that we believe to be intermediates in the cross-linking reaction.

Chromatographic indexes on immobilized artificial membranes for the prediction of transdermal transport of drugs

A set of 12 drugs, consisting of structurally unrelated neutral, basic, acidic and amphoteric compounds, was examined by high performance liquid chromatrography (HPLC) on a model of fluid membrane bilayers, the immobilized artificial membrane (IAM) column. The logarithms of chromatographic capacity factors extrapolated to 100% aqueous phase at pH 5.5 (log k w) were measured and compared to the n-octanol/water partition coefficients (log P). The scale derived from the IAM system was different from the lipophilicity scale expressed by the log P, due to the peculiar capability of phospholipids to well accommodate the ionized form of some molecules and show additive or repulsive extra-interactions when particular structural motifs on the molecule are present. The relationship between log P and log k w previously obtained for compounds interacting on IAM phase by a uniquely lipophilicity-based mechanism, allowed us to calculate, from log P, the values of log k w expected for the drugs considered. These values were subtracted from the log k w experimentally determined and the differences were assumed to quantify the amount of extra-interactions (hydrogen bond and electrostatic interactions) with phospholipids (Δ log k w.). The coefficients of permeability through the human skin ( K p) for the compounds considered did not correlate with either log k w or log P values. However, the K p values correlated well with the Δ log k w values indicating that the higher the ability of a molecule to cross the skin barrier, the lower its component of interaction with phospholipids not accounted for by lipophilicity-based interactions.

A novel solid-state material for furfuraldehyde detection

The thermal degradation of solid insulation within a power transformer results in the production of FFA (2-furfuraldehyde) within the transformer oil. Measuring the concentration of FFA gives a reliable indication as to the condition of the solid insulation. This measurement has routinely been performed by using techniques such as high-performance liquid chromatrography. We report a new solid state material which has been specifically designed for FFA detection. The material can be optically interrogated to detect small concentrations (<1 ppm) of FFA within transformer oil.

Drug assimilation in the tissue of European sea bass (Dicentrarchus labrax) fry delivered orally through bioencapsulation

Assimilation levels of the antibacterials trimethoprim (TMP) and sulfamethoxazole (SMX) in sea bass (Dicentrarchus labrax) fry tissue administered orally were investigated. A 1:5 TMP and SMX combination incorporated in an oil emulsion (Selco) at 20 % and 40 % concentrations (w/w) were bioencapsulated in Artemia (Instar II) nauplii. Chemotherapeutics-loaded ('medicated') nauplii were fed to the sea bass fry and drug concentrations in the tissue were analysed by high-performance liquid chromatrography (HPLC). Fish fed 40 % 'medicated' Artemia assimilated significantly higher levels of chemotherapeutics compared with those fed 20 % 'medicated' Artemia. Chemotherapeutics given at 40 % reached peak levels (19.3 mu g TMP/g DW and 23.31 mu g SMX/g DW) within 2 h while those at 20 % peaked (8.74 mu g TMP/g DW and 6.73 mu g SMX/g DW) after 5 h. TMP persisted in the tissues longer (up to 72 h) than SMX (12-16 h), suggesting a more efficient uptake and retention of the former and/or faster metabolism and elimination of the latter.

Contribution of mass spectrometry to the structural confirmation of components of the antibiotic GE2270 complex.

The GE2270 complex consists mainly of GE2270 A (MW 1289), a thiazolyl peptide antibiotic whose structure originates from the modification of a chain of 14 amino acids in a process which creates six thiazole rings and one pyridine. Together with the main component, a number of structurally related molecules are co-produced in small quantities by fermentation. A preparative high-performance liquid chromatrography method was developed to isolate GE2270 factors B1, B2, C1, C2a, C2b, D1, D2, E and T. Their structures, preliminarily determined by 1H-nuclear magnetic resonance spectroscopy in comparison with GE2270 A, were confirmed by low and high resolution fast-atom bombardment mass spectrometry and studies on the intact molecules and on their main hydrolysis products. Their molecular weights range from 1246 to 1306 Da. The structural differences between the factors lie in the extent of methylation and/or oxidation of thiazole rings (D and E) and asparagine, and in the aromatization of the oxazoline ring.

Quantitative Measurement of Porphyrins in Biological Tissues and Evaluation of Tissue Porphyrins during Toxicant Exposures

Quantitative Measurement of Porphyrins in Biological Tissues and Evaluation of Tissue Porphyrins during Toxicant Exposures. Woods, J. S., and Miller, H. D. (1993). Fundam. Appl. Toxicol. 21, 291-297.Porphyrins are formed in most eukaryotic tissues as intermediates in the biosynthesis of heme. Assessment of changes in tissue porphyrin levels occurring in response to the actions of various drugs or toxicants is potentially useful in the evaluation of chemical exposures and effects. The present paper describes a rapid and sensitive method for the extraction and quantitation of porphyrins in biological tissues which overcomes difficulties encountered in previously described methods, particularly the loss of porphyrins during extraction and interference of porphyrin quantitation by coeluting fluorescent tissue constituents. In this procedure 8- through 2-carboxyl porphyrins are quantitatively extracted from tissue homogenates using HCl and methanol and are subsequently separated from potentially interfering contaminants by sequential methanol/phosphate elution on a C-18 preparatory column. Porphyrins are then separated and measured by reversed-phase high-performance liquid chromatrography and spectrofluormetric techniques. Recovery of tissue porphyrins using this method is close to 100% with an intraassay variability of less than 10%. We have employed this procedure to measure liver and kidney porphyrin concentrations in male Fischer rats and to define the distinctive changes in tissue porphyrin patterns associated with treatment with the hepatic and renal porphyrinogenic chemicals, allylisopropylacetamide, and methyl mercury hydroxide, respectively. This method is applicable to the measurement of tissue porphyrin changes resulting from drug or toxicant exposures in clinical, experimental, or environmental assessments.

Facile separation of the subunits of ovine and bovine gonadotrophins by high-performance liquid chromatrography: microheterogeneity and biological activity

The alpha and beta subunits of ovine and bovine gonadotrophins which undergo instant dissociation in 0.1% (v/v) trifluoroacetic acid in water could be separated into their multiple components by reverse-phase high-performance liquid chromatography on analytical and preparative scales. Several batches of highly purified ovine LH (oLH) separated into four alpha components (alpha 1 to alpha 4) and five beta components (beta 1 to beta 5). While the major alpha component (alpha 3) remained the same in all, the beta subunit was variable in all these preparations of oLH of equal biological and immunoreactivity. Various manipulations, such as prolonged incubation in dilute acid or alkali or conditions prone to air oxidation, did not change elution patterns or produce interconversions among the alpha or beta components, suggesting that microheterogeneity arose in the pituitary itself or during its collection. The subunits obtained on a preparative scale were of high yield and quality and recombined (100%) to produce fully active LH or FSH. Chemical deglycosylations in which 75-80% of the peripheral sugars were removed shifted the elution of all components (alpha as well as beta) slightly but modifications to the protein backbone by other reagents changed the profile of the alpha subunit. Obtaining the various alpha and beta components in high yield would permit a complete analysis of gonadotrophin microheterogeneity.