A quantitative PCR method to quantify ruminant DNA in porcine crude heparin

Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quantitative PCR probe and primer set homologous to the ruminant Bov-A2 short interspersed nuclear element (SINE) locus (Mendoza-Romero et al. J. Food Prot. 67:550–554, 2004) demonstrated nearly equivalent affinities for bovine, ovine, and caprine DNA targets, while exhibiting no cross-reactivity with porcine DNA in the quantitative PCR method. A second PCR primer and probe set, specific for the porcine PRE1 SINE sequence, was also developed to quantify the background porcine DNA level. DNA extraction and purification was not necessary for analysis of the raw heparin samples, although digestion of the sample with heparinase was employed. The method exhibits a quantitation range of 0.3–3,000 ppm ruminant DNA in heparin. Validation parameters of the method included accuracy, repeatability, precision, specificity, range, quantitation limit, and linearity.     

Validation of quantitative polymerase chain reaction methodology for monitoring DNA as a surrogate marker for species material contamination in porcine heparin

Heparin is a widely used intravenous anticoagulant comprising of a very complex mixture of glycosaminoglycan chains, mainly derived from porcine intestinal mucosa. The species of origin and the absence of contaminants from other species are important determinants of the different physicochemical characteristics of heparin. They also determine the potential for introducing infectious and adventitious agents into heparin batches destined for medicinal use. We perform routine quantitative polymerase chain reaction (Q-PCR) release tests to confirm the quality of all crude heparin batches, including those used for the manufacture of enoxaparin sodium. Here we further demonstrate that the assessment of the DNA content in crude heparin is a good surrogate marker of contamination at the mucosa level. After spiking porcine mucosa with ovine mucosa and processing this material to form crude heparin, we were able to observe similar ratios of species-specific DNA in both the starting and end products. Experiments performed with 3,000 and 1,500 ppm contamination found these concentrations to be well above the detection limit for our assay of heparin batches. Additionally this Q-PCR method can be used to detect contamination in mucosa, thus providing a tool capable of monitoring for contaminants throughout the crude heparin manufacturing process. Q-PCR analysis of industrial crude heparin samples has confirmed over time the value of this method to assess the pure porcine origin of heparin.     

Detection of Ovine or Bovine Milk Components in Commercial Camel Milk Powder Using a PCR-Based Method

Food ingredient adulteration, especially the adulteration of milk and dairy products, is one of the important issues of food safety. The large price difference between camel milk powder, ovine, and bovine milk powder may be an incentive for the incorporation of ovine and bovine derived foods in camel milk products. This study evaluated the use of ordinary PCR and real-time PCR for the detection of camel milk powder adulteration based on the presence of ovine and bovine milk components. DNA was extracted from camel, ovine, and bovine milk powder using a deep-processed product column DNA extraction kit. The quality of the extracted DNA was detected by amplifying the target sequence from the mitochondrial Cytb gene, and the extracted DNA was used for the identification of milk powder based on PCR analysis. In addition, PCR-based methods (both ordinary PCR and real-time PCR) were used to detect laboratory adulteration models of milk powder using primers targeting mitochondrial genes. The results show that the ordinary PCR method had better sensitivity and could qualitatively detect ovine and bovine milk components in the range of 1% to 100% in camel milk powder. The commercial camel milk powder was used to verify the practicability of this method. The real-time PCR normalization system has a good exponential correlation (R² = 0.9822 and 0.9923) between ovine or bovine content and Ct ratio (specific/internal reference gene) and allows for the quantitative determination of ovine or bovine milk contents in adulterated camel milk powder samples. Accuracy was effectively validated using simulated adulterated samples, with recoveries ranging from 80% to 110% with a coefficient of variation of less than 7%, exhibiting sufficient parameters of trueness. The ordinary PCR qualitative detection and real-time PCR quantitative detection method established in this study proved to be a specific, sensitive, and effective technology, which is expected to be used for market detection.     

Estimation of the composition of heparin mixtures from various origins using proton nuclear magnetic resonance and multivariate calibration methods

A multivariate calibration method for the characterization of heparin samples based on the analysis of (1)H nuclear magnetic resonance (NMR) spectral data is proposed. Heparin samples under study consisted of two-component or four-component mixtures of heparins from porcine, ovine and bovine mucosae and bovine lung. Although the (1)H NMR spectra of all heparin types were highly overlapping, each origin showed some particular features that could be advantageously used for the quantification of the components. These features mainly concerned the anomeric H, which appeared in the range 5.0-5.7 ppm and the peaks of acetamidomethyl protons at 2.0-2.1 ppm. The determination of the percentage of each heparin class depended on these differences and was carried out using partial least squares regression (PLS) as a calibration method. Prior to the PLS analysis, the spectral data were standardized using the internal standard peak (sodium 4,4-dimethyl-4-silapentanoate- 2,2,3,3- d (4), TSP) as the reference. The quantification of each heparin type in the samples using PLS models built with 4 or 5 components was satisfactory, with an overall prediction error ranging from 3% to 10%.     

Capillary electrophoresis-mass spectrometry - a fast and reliable tool for the monitoring of milk adulteration

The development of a rapid, simple and accurate analytical method aimed at the detection and quantification of bovine milk in either ovine or caprine milk samples by means of CE-MS analyses of whey proteins with high-ionic strength and presence of acidic running buffer is described. The high-ionic strength buffer was used in order to minimize the problems with the adsorption of the proteins onto the fused-silica capillary wall. The acidic running electrolyte, pH 1.9, was used to support the production of positive ions in electrospray. Highly linear dependences of the ratio of the sum of non-bovine beta-lactoglobulins (ovine or caprine) to the total beta-lactoglobulins in milk mixture (bovine plus ovine or bovine plus caprine) vs. the volume percentage of added bovine milk in ovine (or caprine) milk were obtained. This technique allowed the fast and reliable evaluation of milk adulteration. The amount of bovine milk added into the "non-bovine" ones can be well within the concentration range of 5-95%.     

Discontinuous electrophoresis of beta-caseins for the determination of bovine caseins in milk and dairy products.

Discontinuous acidic anodic polyacrylamide gel electrophoresis enables the separation of bovine beta-caseins from those of ovine and caprine. Interfering protein bands as a consequence of ripening or processing have not been detected. After evaluation of the stained gel by laser densitometry, quantification was performed with calibration standards on the same gel by the ratio of the peak areas from bovine to ovine and bovine to caprine, respectively. Thus, independence from the extractability of proteins affected by denaturation and ripening (which might in some cases raise the limit of detection) is achieved. The range of quantification extends from 5 to approximately 70% bovine casein in relation to total casein.     

Bank of reference samples of blank urine from livestock

From three species of livestock, bovine, ovine and porcine, samples of urine from each 10–20 different animals were collected. The animals originated from a governmental experimental farm and differed in race, age, sex and were fed with different types of feed. Fifty different samples of urine were lyophilised in units of 5 mL in sealed amber glass bottles. After lyophilisation of the samples, several quality control tests were performed. The variation in net weight of the units of urine and the lyophilisates ranged from 0.01%–1.7% and 0.1%–10%, respectively. Most values did not exceed 0.1% for urines and 1% for lyophilisates. The residual water content of the lyophilisates was determined by the Karl-Fisher titration method. The average percentage of residual water ranged from 1.0%–7.0% for bovine, from 1.3%–3.0% for porcine and from 1.6%–5.5% for ovine urine lyophilisates. Further, all different samples were analysed for the presence of anabolic compounds with a multi-residue procedure developed at the CRL. In two samples of bovine urine the presence of α-boldenone was detected and confirmed. In most porcine urines the endogenous steroids α-testosterone and α-estradiol were detected. In a number of porcine and ovine urine samples α- and β-zearalenol were detected. The origin of these compounds is the f2-toxin produced by a Fusarium fungus. In ovine samples α-testosterone and α-estradiol were detected.     

Validation and robustness testing of a HPLC method for the determination of avermectins and moxidectin in animal liver samples using an alumina column clean-up

A multi-residue method has been developed for the quantitative determination of moxidectin, abamectin, doramectin and ivermectin in liver samples, with capability for qualitative identification of the presence of eprinomectin. Liver samples are extracted with isooctane, followed by clean-up on alumina-N solid phase extraction (SPE) cartridges. Extracts are derivatised and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery from bovine liver ranged between 90 and 96%. The intra and inter-assay variations showed RSD typically of < 5% and < 10%, respectively. The procedure was applied also to ovine and porcine liver, giving similar results. A robustness study, carried out on the alumina clean-up step, indicated that the step is relatively insensitive to method changes. However, significant differences overall were found for the type of alumina and/or commercial SPE cartridge used. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).     

Studying variations in the PCDD/PCDF profile across various food products using multivariate statistical analysis

Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) are widely recognized by the scientific community as persistent organic pollutants due to their toxicity and adverse effects on wildlife and human health. The actual regulation dedicated to the monitoring of dioxins in food is based on the measurement of 17 congener concentrations. The final result is reported as a toxic equivalent value that takes into account the relative toxicity of each congener. This procedure can minimize the qualitative information available from the abundances of each PCDD/PCDF congener: the characteristic contamination profile of the sample. Multivariate statistical techniques, such as principal component analysis (PCA) or linear discriminant analysis (LDA), represent an interesting way to investigate this qualitative information. Nevertheless, they have only been applied to the analysis of contamination data from food products and biological matrices infrequently. The objective of the present study was to analyze a large data set from dioxin analyses performed on various food products of animal origin. The results demonstrate the existence of differences in congener-specific patterns between the analyzed samples. Variability was first demonstrated in terms of the food type (fish, meat, milk, fatty products). Then a variability was observed that was related to the specific animal species for meat and milk samples (bovine, ovine, porcine, caprine and poultry). Some practical applications of these results are discussed. The origin(s) of the observed differences, as well as their significance, now remain to be investigated, both in terms of environmental factors and transfer through living organisms. A better knowledge of the relation between a contamination profile and its specific source and/or food product should be of great interest to scientists working in the fields of contaminant analysis, toxicology and metabolism, as well as to regulatory bodies and risk assessors in charge of final decisions regarding the eventual hazards associated with theses substances.     

New developments in quantitative polymerase chain reaction applied to control the quality of heparins

Heparin is a widely used intravenous anticoagulant comprised of a very complex mixture of glucosaminoglycan chains, mainly derived from porcine intestinal mucosa. Recent contamination of heparin with oversulfated (OS) chondroitin sulfate resulted in a significant number of deaths, triggering a rapid revision of product monographs and the introduction of new analytical methods to limit as far as possible the chances of another occurrence of such a phenomenon. The distribution of heparin-processing units across the globe prevents their complete fool-proof auditing. Therefore, the implementation of additional orthogonal analytical techniques for quality control (QC) of heparin batches is highly important. We perform routine quantitative polymerase chain reaction (Q-PCR) release tests to confirm the quality of all crude heparin batches received by sanofi-aventis. The routine test used provides information on the animal species of origin as requested by the US Pharmacopoeia (USP) and European Pharmacopoiea monographs. Here, we demonstrate that the Q-PCR test is inhibited by OS glycosaminoglycans at concentrations as low as 0.5% (w/w versus heparin) and can be used as an additional safeguard to monitor levels of potentially harmful contaminants without any increased workload. In response to a request from the USP, we also describe the development of a Q-PCR method for monitoring nucleotidic impurities in pure heparin, which is able to detect amplifiable DNA at concentrations lower than 0.1 ng DNA per milligram of heparin. This increased sensitivity makes this modified Q-PCR method a potential candidate for inclusion as a QC requirement in future monographs.     

Specific Non-Reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid

Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in N-sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.     

Isolation and identification of O- and N-linked glycoproteins in milk from different mammalian species and their roles in biological pathways which support infant growth

Milk serves as the sole nutrition for newborns, as well as a medium for the transfer of immunological components from the mother to the baby. This study reveals different glycoprotein profiles obtained from human, bovine, and caprine milk and their potential roles in supporting infant growth. Proteins from these three milk samples are separated and analyzed using two-dimensional gel electrophoresis (2-DE). Glycosylated proteins from all samples are enriched by affinity chromatography using lectins from the seeds of Artocarpus integer before analysis using LC/MS-QTOF. The glycoproteome profiling demonstrates that glycosylated proteins are higher in caprine milk compared to other samples. Analysis using LC/MS-QTOF identified 42 O-glycosylated and 56 N-glycosylated proteins, respectively. Among those identified, human milk has 17 glycoproteins, which are both O- and N-glycosylated, whereas caprine and bovine have 10 and 1, respectively. Only glycoproteins from human milk have shown positive matching to important human biological pathways, such as vesicle-mediated transport, immune system and hemostasis pathways. Human milk remains unique for human babies with the presence of antibodies in the form of immunoglobulins that are lacking in ruminant milk proteomes.     

Determination of processed animal proteins, including meat and bone meal, in animal feed

An intercomparison study was conducted to determine the presence of processed animal proteins (PAPs), including meat and bone meal (MBM) from various species, in animal feed. The performances of different methods, such as microscopy, polymerase chain reaction (PCR), immunoassays, and a protocol based on iquid chromatography (LC), were compared. Laboratories were asked to analyze for PAPs from all terrestrial animals and fish (total PAPs); mammalian PAPs; ruminant PAPs; and porcine PAPs. They were free to use their method of choice. In addition, laboratories using microscopy were asked to determine the presence of PAPs from terrestrial animals, which is applicable only to microscopy. For total PAPs microscopy, LC and some immunoassays showed sufficient results at a concentration as low as 0.1% MBM in the feed. In contrast, PCR was not fit for purpose. In differentiating between MBM from terrestrial animals and fishmeal, microscopy detected 0.5% of terrestrial MBM in feed in the presence of 5% fishmeal, but was less successful when the concentration of MBM from terrestrial animals was 0.1%. The animal-specific determination of MBM from mammals or, more specifically from either ruminants or pigs, by PCR showed poor results, as indicated by a high number of false-positive and false-negative results. The only PCR method that scored quite well was applied by a member of the organizer team of the study. Immunoassays scored much better than PCR, showing sufficient sensitivity but some deficiency in terms of specificity. The results also demonstrated that the reliable determination of MBM from ruminants has not been resolved, especially for low concentrations of MBM (0.1%) in feed. Comparison of the results for mammalian MBM from all methods indicated that, for control purposes, the immunoassay method, especially when applied as dipsticks, could be used as a rapid screening method combined with microscopy to confirm the positive samples. However, implementation of such a system would require that the immunoassays were previously validated to demonstrate that this approach is fit for purpose. The determination of ruminant or porcine PAPs by immunoassays was more difficult, partly because the MBM in this study contained about 50% bovine and porcine material, thereby reducing the target concentration level to 0.05%.     

Analysis and characterization of heparin impurities

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007?C2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations.

Polymerase chain reaction method to detect canis materials by amplification of species-specific DNA fragment

Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.     

A novel, sensitive and specific real-time PCR for the detection of traces of allergenic Brazil nut (Bertholletia excelsa) in processed foods

Food-allergic individuals have to strictly avoid the offending food because no causative immunotherapies are available. Thus, reliable labelling of allergenic ingredients or precautionary labelling of cross-contacts with potential allergens is of major importance. Verification of compliance with labelling requirements and identification of cross-contacts demand test methods that enable the specific and sensitive detection of the analyte. Brazil nut (Bertholletia excelsa) is such a food commodity with allergenic potential. We describe the development of a novel qualitative real-time polymerase chain reaction (PCR) specific for Brazil nut DNA and its comparison with a qualitative commercially available lateral flow device (LFD) that detects Brazil nut protein. Specificity was investigated with 58 foods, and no false-positive reactions were observed in real-time PCR. The sensitivity was investigated with spiked chocolate and incurred dough samples as well as cookies baked thereof. The simultaneous spiking of matrices with identical amounts of Brazil nut and peanut between 5 and 100,000 mg/kg allowed the verification of the spike quality with two peanut-specific enzyme-linked immunosorbent assay. The real-time PCR detected Brazil nut in all three matrices down to the lowest investigated spike level of 5 mg/kg. The real-time PCR results from the analysis of 15 retail samples were confirmed by LFD results and were in concordance with the labelling of products. The real-time PCR showed unparalleled specificity, and primary data indicated potentially quantitative features in spiked and retail samples. Because of entirely reproducible chemistry of this real-time PCR, this is the first generally available Brazil nut-specific detection method with an appropriate sensitivity to help avoid severe allergic reactions for Brazil nut-allergic individuals.     

Electrophoresis for the analysis of heparin purity and quality

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment.     

Determination of twelve heparin- and heparan sulfate-derived disaccharides as 2-aminoacridone derivatives by capillary zone electrophoresis using ultraviolet and laser-induced fluorescence detection

In quest for high sensitivities, we developed an ultrahigh capillary electrophoresis (CE) method for the structural analysis of heparin and heparan sulfate (HS) in biologic samples. Heparin and HS were digested with an equi-unit mixture of heparin lyases I, II and III and the obtained Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone. All known twelve non-, mono-, di- and trisulfated Delta-disaccharides were completely resolved in a single run, using 50 mM phosphate buffer, pH 3.5, and reversed polarity at 30 kV. Relative standard deviation in migration times and peak areas as well as day-to-day variance ranged from 0.9 to 2.4%, suggesting a reproducible and precise method. Detection of 2-aminoacridone (AMAC)-derivatives of Delta-disaccharides by UV at 255 nm showed 2.8 and 10 times higher sensitivity than that of derivatized and non-derivatized ones at 232 nm. Laser-induced fluorescence detection with an Ar-ion laser source showed an approximately 100 times higher sensitivity than that obtained at 232 nm of the non-derivatized species. Application of this method to quantitative analysis of Delta-disaccharides derived from porcine intestinal mucosa heparin and bovine kidney HS showed excellent agreement with previously published methods, suggesting an accurate method. The developed method can be easily applied for the disaccharide analysis of heparin/HS at the attomole level with high accuracy, for distinguishing between heparin and HS and may be of value for studying their interactions with matrix effective molecules.     

High-sensitivity visualisation of contaminants in heparin samples by spectral filtering of 1H NMR spectra

A novel application of two-dimensional correlation analysis has been employed to filter (1)H NMR heparin spectra distinguishing acceptable natural variation and the presence of foreign species. Analysis of contaminated heparin samples, compared to a dataset of accepted heparin samples using two-dimensional correlation spectroscopic analysis of their 1-dimensional (1)H NMR spectra, allowed the spectral features of contaminants to be recovered with high sensitivity, without having to resort to more complicated NMR experiments. Contaminants, which exhibited features distinct from those of heparin and those with features normally hidden within the spectral mass of heparin could be distinguished readily. A heparin sample which had been pre-mixed with a known contaminant, oversulfated chondroitin sulfate (OSCS), was tested against the heparin reference library. It was possible to recover the (1)H NMR spectrum of the OSCS component through difference 2D-COS power spectrum analysis of as little as 0.25% (w/w) with ease, and of 2% (w/w) for more challenging contaminants, whose NMR signals fell under those of heparin. The approach shows great promise for the quality control of heparin and provides the basis for greatly improved regulatory control for the analysis of heparin, as well as other intrinsically heterogeneous and varied products.     

Determination of galactosamine impurities in heparin samples by multivariate regression analysis of their <Superscript>1</Superscript>H NMR spectra

Heparin, a widely used anticoagulant primarily extracted from animal sources, contains varying amounts of galactosamine impurities. Currently, the United States Pharmacopeia (USP) monograph for heparin purity specifies that the weight percent of galactosamine (%Gal) may not exceed 1%. In the present study, multivariate regression (MVR) analysis of ¹H NMR spectral data obtained from heparin samples was employed to build quantitative models for the prediction of %Gal. MVR analysis was conducted using four separate methods: multiple linear regression, ridge regression, partial least squares regression, and support vector regression (SVR). Genetic algorithms and stepwise selection methods were applied for variable selection. In each case, two separate prediction models were constructed: a global model based on dataset A which contained the full range (0–10%) of galactosamine in the samples and a local model based on the subset dataset B for which the galactosamine level (0–2%) spanned the 1% USP limit. All four regression methods performed equally well for dataset A with low prediction errors under optimal conditions, whereas SVR was clearly superior among the four methods for dataset B. The results from this study show that ¹H NMR spectroscopy, already a USP requirement for the screening of contaminants in heparin, may offer utility as a rapid method for quantitative determination of %Gal in heparin samples when used in conjunction with MVR approaches.