Title of Invention	METHOD OF DETERMINING SPECIFIC GROUPS OF FORMING HEPARINS
Abstract	The Present invention relates to a method of analysing heparins or low-molecular-weight heparins. The inventive method is characterised in that the sample to be dosed is depolymerised by heparinases and, if necessary, the depolymerisate obtained is reduced. A high-performance liquid chromatography analysis is then performed.

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Method of determining specific groups forming heparins or low-molecular-weight heparins The subject of the present invention is a method for analysing specific groups constituting heparins or low-molecular-weight heparins. During the process for preparing enoxaparin (Lovenox®) (US5,389,618) from pure heparin, the aqueous-phase alkaline depolymerization process produces a partial but characteristic conversion of the glucosamines of the reducing ends of the oligosaccharide chains. The first step of this conversion consists of a glucosaminemannosamine epimerization (T. Toida et al., J. Carbohydrate Chemistry, 15(3), 351-360 (1996)); the second step is a 6-0-desulfation of the glucosamine, leading to the formation of derivatives called "1,6 anhydro" (international patent application WOOl/29055) . This type of derivative is only obtained for oligosaccharide chains whose terminal glucosamine is 6-0-sulfated. The percentage of oligosaccharide chains whose end is modified with a 1,6-anhydro bond is a structural characteristic of the oligosaccharide mixture of Lovenox and it should be possible to measure it. The present invention therefore consists of a method for analyzing heparins, low-molecular-weight heparins and more particularly Lovenox. The method of analysis according to the invention is the following: The sample to be assayed is depolymerized by the action of heparinases and then, where appropriate, the depolymerizate obtained is reduced and then analysis is carried out by high-performance liquid chromatography. The method as defined above is therefore characterized in that there is a search for the presence of oligosaccharide chains whose end is modified with a 1,6-anhydro bond ("1,6-anhydro groups"). In particular, the sample to be assayed is first of all exhaustively depolymerized with a mixture of heparinases and in particular heparinase 1 (EC 4.2.2.7.), heparinase 2 (heparin lyase II) and heparinase 3 (EC4.2.2.8.)). (These enzymes are marketed by the group Grampian Enzymes). International application WO88/02400 describes a method of depolymerizing heparin by a heparinase and neutralizing the anticoagulant activity of the heparin contained in a blood sample. The subject of the invention is therefore a method for analyzing heparins or low-molecular-weight heparins, characterized in that the following steps are carried out: 1) depolymerization of the sample by the action of heparinases 2) where appropriate, reduction of the depolymerizate 3) assay by high-performance liquid chromatography. The subject of the invention is more particularly the method as defined above, characterized in that the heparinases are in the form of a mixture of heparinase 1 (EC 4.2.2.7.), heparinase 2 (heparin lyase II) and heparinase 3 (EC.4.2.2.8.). The depolymerizate thus prepared is then treated preferably ".vith an Na3H4 sclution in scdium acetate. The latter cceraticn makes it possible to specifically reduce the reducing ends which are not in the 1,6-anhydro form ^prcducts described in patent application /."O"01/72762) . rinally, in order to he able to quantify the disacchariies 1 and 2 described below, the sam.ple of lc".v-molecular--..-3i5ht heparin, depolymerized with heparinases, anould be reduced by the accion of a reducing agent such as NaBH4- therefore more The subject cf particularly z"r.e in that the cocci cne invention is ichcd as defined above, characterized .ericed heparin is then reduced. The subject cf the invention is miost particularly the m>ethod " as defined above, characterized in that the reducing agent is ".^aBH^. Another alkali mietal salt of borchydride such as lithium or potassium m,ay be optionally used. The assay of z"r.e l,6-3nhydro ends is then carried out by ."i?LC (High Ferfcrm.ance Liquid Chrcm.atography) and in particular by ana ^".-a;;ahange chrcmiatcgraphy. ct: cssible to : r low-r:cl ccntain these the method of issay according to the invention m.akes it :learly differentiate Lcvenox from the esular-v;eight heparins "which do not "1,c-anhydro" derivatives. Conversely, issay according to the in"/ention miakes it possible to ascertain that Icw-molecular-weight heparins do not satisfy the physicocherrdcal characteristics of Lcvenox and therefore are different in "nature. The method of assay according to the invention ~ay be applied to the industrial process during in-process control of samples in order to provide standardization of the process for ~anufacturing Lovenox and to obtain uniform batches. After enzymatic depolymerization and reduction of the reducing ends, the 1,6-anhydro derivatives of Lovenox exist in 4 essential forms. The subject of the invention is therefore also the method as described above, characterized in that the 1,6-anhydro residues obtained during the depolymerization reaction are the following: All the oligosaccharides or polysaccharides v."hich contain the 1,6-anhydro end on the terminal disaccharide unit and which do not possess a 2-0-sulfate on the uronic acid of said terminal disaccharide are ocr.pletely depolymerized by the heparinases and in the form of the disaccharides 1 and 2. On the o"ther har.d, when said terminal saccharide contains a 2-0-sulfate on the uronic acid and when it is in the manncsamir.e form, the 1,6-anhydro derivative is in the form of the tetrasaccharide 1 (form resistant to heparinases). The trisaccharide 1 (see below) is also present in the mixture. It is derived from another degradation process which leads to the structure below (peeling phenomenon observed during the chemical depolymerization of Lovenox). The other constituents of the mixture are not characteristic solely of Lovenox. There are of course the 8 elementary disaccharides of "the heparin chain. These 8 elementary disaccharides are marketed inter alia by the company Sigma. Other disaccharides were identified in the mixture by the method according to the invention: the disaccharides AllSgai and AlVSgai which have as origin alkaline 2-0-desulfation of -IdoA(2S)-GlcNS(6S)- and of -IdoA(2S)-GlcNS-, leading to the formation of 2 galacturonic acids. They are not usually present in the original structure of heparin (U.M. Desai et al., Arch. Biochem. Biophys., 306 (2) 461-468 (1993). The oligosaccharides containing 3-0-sulfated glucosamines withstand cleavage by heparinases- and remain present in the form of tetrasaccharides. In the case of most low-molecular-weight heparins, the heparin is extracted from pig mucus, and these principal tetrasaccharides are represented below. They are resistant to enzymatic depolymerization and reflect the sequences with affinity for antithrombin III. They are syiribolized as follows: Alla-IISgiu and Alla-IVSgiu. (S. YAMADA, K. YCSHIDA, M. SUGIURA, K-H KHOO, H.R. MORRIS, A. DELL, J. Biol. Chem.; 270(7), 4780-4787 (1993) The final constituent of the mixture cleaved with heparinases is the glycoserine end AGlcA-Gal-Gal-Xyl-Ser (K. SUGAHARA, H. TSUDA, K. YOSHIDA, S. YAT-IADA, J. Biol. Chem.; 270(39), 22914-22923 (1995); K. SUGAKARA, S. YAKADA, K. YOSHIDA, P. de WAARD, J.F.G. VLIEGENTHART; J.Biol.Chem.; 267(3), 1528-1533 (1992). The latter is generally almost absent from Lovenox (see NMR in Example" 5). Another aspect of the invention consists in the chromatography process used for determining the 1,6-anhydro groups. First of all, it involves separating the various polysaccharides obtained after depolymerization and treatment with a reducing agent such as NaBH4. Anion-exchange chrcmatcgraphy (SAX) is the separating method which is most suitable for such a complex mixture. Columns filled with a stationary phase of the Spherisorb SAX type having a particle size of 5 pm and a length of 25 cm can be used. All the conventicnal column diameters between 1 mm and 4.6 mm can be used. The equipment used may be a chromatograph allowing the formation of an elution gradient with a UV detector, more preferably equipped with an array of diodes in order to be able to produce UV spectra of the constituents and to record complex signals, resulting from the difference between the absorbance at 2 different wavelengths and allowing the specific detection of acetylated oligosaccharides. To allow this type of detection, mobile phases which are transparent in the UV region up to 200 nm are preferable. This excludes conventional mobile phases based on NaCl which have moreover the" disadvantage of requiring a passivated chromatcgram in order to withstand the corrosive power of the chlorides. The mobile phase used here will be preferably based on sodium perchlorate, but methanesulfonate or phosphate salts may also be used. The pH recommended for the separation is from 2 to 6.5. Preferably, a pH in the region of 3 will be used. It is controlled here by adding a salt such as phosphate possessing a buffering power at pH = 3 which is better than that of perchlorates. By way of example, standard chromatographic separation conditions are given below: Solvent A: NaHaPO^, 2.5 mM, brought to pH 2.9 by addition of H3PO4 Solvent B: NaC104 IN- :iaH2P04, 2.5 mM, brought to pH 3.0 by addition of H3PO4 The elution gradient may be the following: T = 0 min: %B = 3; T = 40 min: IB = 60; T = 60 min: %B = 80 The subject of the present invention is therefore also a method of analysis as defined above by separation by anion-exchange chromatography, characterized in that the mobile phase which is transparent in the UV region up to 200 nM is used. The subject of the invention is more particularly a mobile phase as defined above based on sodium perchlorate, methanesulfcnate salts or phosphate salts. Another most important aspect consists in the method of detection. A method is developed in order to increase the specificity of the UV detection. As nonacetylated polysaccharides all have, at a given pH, a fairly similar UV spectrum, it is possible to selectively detect the acetylated sugars ..by taking as signal the difference between the absorbance at 2 wavelengths chosen such that the absorptivity of the nonacetylated saccharides cancels out. In the case below, 202 nm and 230 nm will be chosen as detection and reference wavelengths and the 202-230 nm signal will be noted. The choice of course depends on the pH of the mobile phase (adjustments of a few nm may be necessary so as to be at the optimum of said conditions) . The rr.cst suitable detector for this technique is the DAD 1100 detector from the company Agilent Technologies. In this case, a double detection will be carried cut at 234 r.m, on the one hand, and at 202-230 nm, on the. other hand. The principle of "selective detection of acetylated oligosaccharides is illustrated in the figure below in which the UV spectrum of a sulfated disaccharide Delta Is is compared with that of an acetylated disaccharide Delta la. The subject of the present invention is therefore also a method of analysis as defined above by separation by anion-exchange chrc-atography, characterized in that the method of detection makes it possible to selectively detect acetylated sugars.. The subject of the invention is also most particularly a method of analysis as defined above by separation by exchange chromatography, characterized in that the selective detection of acetylated sugars is carried out taking as signal the difference between the absorbance at 2 wavelengths chosen such that the absorptivity of the nonacetylated saccharides cancels out. The quantification of the 4 1,6-anhydro residues described above req-^ires a sufficient selectivity of the chrcmatcgraphic system in relation to all the ether constituents of the ~ixture. However, the 2 disaccharides 1 and 2, which are coeluted in general, are poorly resolved with respect to Alia, especially as the latter is present in the form of its 2 a and P anomers. The identity of the 2 disaccharides 1 and 2 may be easily verified because they form in a few hours at room temperature in an aqueous solution of Alls brought to pH 13 by addition of NaOH. However, if double detection is used, the acetylated oligosaccharides AlVa, Alia, Villa, Ala, Vlla-IVSqiu and Alla-IISqiu are easily identifiable. The- causes of splitting of the peaks are the anomeric forms, on the one hand, and to a lesser degree the glucosamine *-* manncsamine epimerization which is partially present for Alls, AIIIs and Als when they are in the terminal position in the oligosaccharide chain. In order to be able to quantify the disaccharides 1 and 2, the sample of low-molecular-weight heparin, depolymerized by heparinases is reduced by the action of NaBH4. This reduction has the advantage of eliminating the a The examples of chromatograms described in Figures 1 and 2 belcw clearly illustrate these phenomena and the advantages of this method. Finally, the subject of the invention is also the novel saccharide derivatives obtained using the depolymerization and reduction process, chosen from disaccharide 1, disaccharide 2, disaccharide 3 and trisaccharide 1. The examples below illustrate the invention without however having a limiting character. Example 1: The enzymatic depolymerization is carried out for 48 hours at room temperature by mixing 50 pi of a solution containing 20 mg/ml of low-molecular weight heparin to be assayed, 200 pi of a 100 mM acetic acid/NaOH solution at pH 7.0 containing 2 mM calcium acetate and 1 mg/ml of BSA with -50 pi of the stock solution of the 3 heparinases. The reduction is carried out on 60 pi of the product depolymerized with the heparinases by adding 10 pi of an NaBH4 solution at 30 g/1 in 100 mM sodium acetate prepared immediately before use. It will be noted that the heparinases are stored at -30°C. The heparinases are in a buffer solution and their titer is 0.5 lU/ml (composition of the buffer solution: aqueous solution pH 7 of KH2PO4 at a concentration of 0.01 mol/1 and supplemented with bovine serum albumin (BSA) at 2 mg/ml). Example 2: . . NMR of Disaccharide 3 obtained according to the process described above. Proton spectrum in D^O, 400 MHz, T=298K, 5 in ppm: 3.34 (IH, dd, J=7 and 2Hz, H2) , 3.72 (IH, t, J=8Hz, H6) , 3.90 (IH, m, H3), 4.03 (IH, s, H4), 4.20 (IH, d, J=SHz, H6), 4.23 (IH, t, J=5Hz, H3") , 4.58 (IH, m, H2"), 4.78 (IH, m, H5), 5.50 (IH, s, HI), 5.60 (IH, dd, J=6 and IHz, HI"), 6.03 (IH, d, J=5Hz, H4")]. Example 3 NMR of the Tetrasaccharide 1 obtained according to the process described above. Proton spectrum in D2O, 400 MHz, T=298K, 5 in ppm: 3.15 (IH, s, H2), 3.25 (IH, m, H2"), 3.60 (IH, m, H3" ) , between 3.70 and 4.70 (14H, unresolved coiriplex, H3/H4/H6, H2"/H3"/H4"/H5", H4"/H5"/H6" , H2""/H3""), 4.75 (IH, m, H5) , between 5.20 and 5.40 (2H, m, HI" and HI"), 5.45 (IH,* m, HI""), 5.56 (IH, m, HI), 5.94 (IH, d, J=5Hz, H4) Example 4: NMR of the Trisaccharide 1 obtained according to the process described above. Spectrum in D2O, 600 MHz, (5 in ppm): 3.28 (IH, m) , 3.61 (IH, t, 7Hz), 3.79 (IH, t, 7Hz) , 3.95 (IH, d, 6Hz) , 4.00 (IH, s), 4.20 (IH, m), 4.28 (2H, m) , 4.32 (IH, d, 4Hz), 4.41 (IH, s), 4.58 (IH, s) , 4.61 (IH, s) , 4.90 (IH, broad s), 5.24 (IH, s) , 5.45 (IH, s), 5.95 (IH, s) . Example 5: NMR of AGlcA-Gal-Gal-Xyl-Ser Spectrum in D^O, 500 MHz (5 in ppm): 3.30 (IH, t, 7Hz) , 3.34 (IH, t, 8Hz), 3.55 (IH, t, 7Hz) , 3.60 (IH, t, 7Hz), between 3.63 and 3.85 (lOH, m) , 3.91 (2H, m) , , • 3.96 (IH, dd, 7 and 2Hz) , between 4.02 and 4.10 (3H, m), 4.12 (IH, d, 2H2), 4.18 (IH, m) , 4.40 (IH, d, GHz), 4.46 (IH, d, 6Hz), 4.51 (IH, d, 6Hz) , 5.29 (IH, d, 3Hz), 5.85 (IH, d, 3Hz) . Example 6 : Principle of the qriantification In the method according to the invention, the v;idely accepted hypothesis that all the unsaturated oligosaccharides contained in the raixture have the same molar absorptivity, equal to 5500 mol"-^. 1. cm"-^ is rr.ade. It is therefore possible to determine the percentage by weight of all the constituents of the depolymerized mixture in the starting low-molecular-weight heparin. For the 4 1,6-anhydro derivatives which correspond to the peaks 7,8,13 and 19, the following percentages by weight are obtained: Area?, Areas, Areai3 and Areai9 correspond to the areas ) of each of the peaks 7, 8, 13 and 19. The molar masses of each of these 4 cc-pounds are 443, 443, 545 and 1210 respectively. 2^ ^^x ""^^^^x corresponds to the ratio of the area of each peak of the chromatogram by the molar mass of the corresponding product. If M„ is the mean r.ass of the low-molecular-weight heparin studied, the percentage of oligosaccharide chains ending v.-ith a 1,6-anhydro ring is obtained in the following manner: The molecular masses of the constituents are the following: - - Nomenclature of the saccharides and correspondence with the peaks according to Figures 1 and 2 IdoA: a-L-Idopyranosyluronic acid; GlcA: p-D-Glucopyranosyluronic acid; AGlcA: 4,5-unsaturated acid: 4-deoxy-a-L- tiirec?]exenepyrancsyluronic acid; Gal: D-Galactose; Xyl: xylose; GlcNAc: 2-dec;-:y-2-acetamido-a-D-glucopyranose; GlcNS: 2-decxy-2-£ulfaraido-a-D-glucopyranose; 2S:. 2-0-£ulfate, 3S: 3-0-s"jlfate, 6S: 6-0-£ulfate 1: AGlc.-.3i-3 Gal P1-3 Galpi-4 Xyl pl-O-Ser 4-decxy-a-L-threohexenepyranosyluronic acid-(l->4)-2-deoxy-2-acetamido-a-D-glucopyrancsyl sodium salt AGlc?.P:-3 Gal P1-3 Galpi-^ Xyl pi-O-CHz-COOH 4-deo>:y-a-L-threchex-4-enegalactopyranosyl-urcnic acid- (l->4) -2-deoxy-2-sulfamido-P-D-glucopyranose disodium salt 4-deoKy-a-L-threohexenepyranosyluronic acid-(1-^4)-2-decxy-2-sulfamido-a-D-glucopyrancsyl sodium salt 4-deoxy-a-L-threohexenepyranosyluronic acid-(l->4)-2-deoxy-2-acetamido-6-0-sulfo-a-D-gluco-pyrancsyl disodium salt 4-deoxy-a-L-threohex-4-enepyranosyluronic acid-(l->4) -1, 6-anhycifo-2-deoxy-2-sulfamido-P-D-glucopyranose disodium salt (disaccharide 1) 4-deoxy-a-L-threohex-4-enepyranosyluronic acid-{1-^A)-1,6-anhydro-2-deoxy-2-sulfamido-P-D-mannopyranose disodium salt (disaccharide 2) 4-deoxy-2-0-sulfo-a-L-threohexenepyranosyl-uronic acid- (l->4 ) -2-deoxy-2-acetamido-a-D-glucopyranosyl disodium salt 4-decxy-a-L-threohex-4-enegalactopyranosyl-uronic acid- (l->4) -2-deoxy-2-sulf amido-6-0-sulfo-p-D-glucopyranose trisodium salt 4-decxy-a-L-threohexenepyranosyluronic acid-(1-^4)-2-decxy-2-sulfamido-6-0-sulfo-P-D-gluccpyranosyl trisodium salt 4-decxy-2-0-sulfo-a-L-threchexenepyranosyl-uronic acid- (l->4 ) -2-deoxy-2-sulfamido-a-D-cluccpyranosyl trisodium salt 4-decxy-2-0-sulfo-a-L-threchex-4-enepy.rano£yl-uronic acid-(l->4)-l,6-anhydro-2-decxy-2-sulfamido-P-D-glucopyrancse trisodium salt (Disaccharide 3) 4-deoxy-2-0-sulfo-a-L-thr£chexenepyranosyl-urcnic acid- (l->4) -2-deoxy-2-acetan\ido-6-0-sulfo-a-D-glucopyranosyl trisodium salt 4-deoxy-a-L-threGhexenepyranosyluronic acid-(l->4)-2-decxy-2-acetamido-6-0-sulfo-a-D-gluco-pyranosyl-(1-^4) -p-D-gluccpyranosyluronic acid-(1-^4) -2-decxy-2-sulfainido-3-0-sulfo-a-D-gluco-pyranosyl) pentasodium salt 4-deoxy-2-0-sulfo-a^L-threohexenepyranosyl-uronic acid-(l->4 ) -2-deoxy-2-sulfamido-6-0-sulfo-a-D-glucopyranosyl tetrasodium salt 4-deoxy-a-L-threohexenepyranosyluronic acid-(1—>4)-2-deoxy-2-acetamido-6-0-sulfo-a-D-gluco-pyranosyl- (1-^4) -p-D-glucopyranosyluronic acid-(1-^4) -2-deoxy-2-sulfaiuido-3, 6-di-O-sulfo-a-D-glucopyranosyl) hexasodium salt 4-deoxy-2-0-sulfo-a-L-threohexenepyranosyl-uronic acid-(l->4) -2-deoxy-2-sulfamido-6-0-sulfo-D-glucopyranosyl- {l->4) -2-0-sulfo-a-L-idopyranosyluronic acid hexasodium salt 4-deoxy-2-0-sulfo-a-L-threohexenepyranosyl-uronic acid-(l->4 )-2-deoxy-2-sulfamido-6-0-sulfo-a-D-glucopyranosyl- (l->4) -2-0-sulfo-a-L-idopyranosyluronic acid-(l->4) -1, 6-anhydro-2-deoxysulfamido-p-D-mannopyranose, hexasodium salt (tetrasaccharide 1) 4-deoxy-a-L-threohexenepyranosyluronic acid-(l->4)-2-decxy-2-acetamido-a-D-glucitol sodium salt 4-deoxy-a-L-threohexenepyranosyluronic acid-(l->4)-2-decxy-2-sulfamido-p-D-glucitol disodium salt 4-decxy-a-L-threohexenepyranosyluronic acid-(l->4) -2-decxy-2-sulf $raido-a-D-glucitol "disodium salt 4-deoxy-a-L-threGhexenepyranosyluronic acid-(l-->4) -2-decxy-2-acetamido-6-0-sulfo-a-D-glucitol disodium salt 4-decxy-2-0-sulfo-a-L-threohexenepyranosyl-urcnic acid- (1-^4) -2-deoxy-2-acetamido-a-D-glucitol disodium salt 4-deoxy-a-L-threohexenegalactopyranosyluronic acid- (l->4)-2-deoxy-2-sulfamido-6-0-sulfo-P-D-glucitol trisodium salt 4-deoxy-a-L-threohexenepyranosyluronic acid-(1-^4)-2-decxy-2-sulfamido-6-0-sulfo-a-D-glucitol trisodium salt 4-deoxy-2-0-sulfo-a-L-threohexenepyranosyl-urcnic acid- (1-^4) -2-deoxy-2-sulf amido-a-D-glucitol trisodium salt 4-deoxy-2-0-sulfo-a-L-threohexenepyranosyl-uronic acid-(l->4)-2-deoxy-2-acetamido-6-0-sulfo-a-D-glucitol trisodium salt 4-deoxy-a-L-threohexenepyranosyluronic acid-(1-^4)-2-decxy-2-acetamido-6-0-sulfo-a-D-gluco-pyranosyl- (l->4) -p-D-glucopyranosyluronic acid-(1-^4)-2-deoxy-2-sulfamido-3-0-sulfo-a-D-glucitol) pentasodium salt 4-deoxy-2-0-sulfo-a-L-threohexenepyranosyl-uronic acid-(1-^4 )-2-deoxy-2-sulfamido— 6-0-sulfo-a-D-glucitol trisodium salt 4-deoxy-a-L-threohexenepyranosyluronic acid-(l->4)-2-deoxy-2-acetamido-6-0-sulfo-a-D-glucopyranosyl- (l->4) -p-D-glucopyranosyl-uronic acid- (l->4)-2-deoxy-2-sulfamido-3,6-di-0-sulfo-a-D-glucitol) hexasodium salt 4-decxy-2-0-sulfo-a-L-threohexenepyranosyl-uronic acid-(l->4)-2-deoxy-2-sulfamido-6-0-sulfo-a-D-glucopyranosyl-(1-^4)-2-0-sulfo-a-L-idopyranosyluronic acid hexasodium salt (form reduced with NaBH4) . Figure 1 Chromatographic separation of enoxaparine depolymerized v;ith heparinases before and after reduction with :?aBH4 (signal in fine black: UV at 234 nm; signal in thick black: UV at 202-234 nm) Figure 2: Chrc.Tiatographic separation of heparin depolyraerized with heparinases before and after reduction with IJaEH4 (signal in fine black: UV at 234 nm; signal in thick black: UV at 202-234 nm) WE CLAIM; 1. A method for analysing heparins or low-molecular weight heparins to carry out a search for the presence of oligosaccharide chains whose end is modified with a 1,6- anhydro bond, characterized in that the following steps are carried out: 1 - depolymerization of the sample by the action of heparinases 2 - reduction of the depolymerizate 3 - assay by high-performance liquid chromatography, the chromatographic method used being an anion-exchange chromatography, in which a mobile phase which is transparent in the UV region up to 200Nmis preferably used, and in which a method of detection which makes it possible to selectively detect acetylated sugars is preferably used, said method being carried out taking as signal the difference between the absorbance at two wavelengths chosen such that the absorptivity of the nonacetylated saccharides cancels out. 2. The method as claimed in claim 1, wherein the heparinases are in the form of a mixture of heparinase 1 (EC 4.2.2.7), haprinase 2 (heparin lyase II) and haparinase 3 (EC.4.2.2.8.). 3. The method as claimed in claim 1, wherein the heparin depolymerized by the action of heparinase (depolymerizate) is then subjected to a reducing agent. 4. The method as claimed in claim 3, wherein the reducing agent is NaBH4 or an alkali metal salt of the borchydride anion. 5. The method as claimed in claim 1, wherein the mobile phase used is based on sodium perchlorate, methanesulfonate salts or phosphate salts. 6. The method as claimed in claim 1, wherein the 1, 6-anhydro residues obtained during the depolymeriztion reaction are the following : 7. 8. A 1,6-anhydro derivative of formula (disaccharide 2) 9. A 1,6-anhydro derivative of formula (disaccharide 3) 10. A trisaccharide derivative of formula:

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