LC-ms / ms-based method for quantifying hyaluronic acid, BDDE and BDDE cross-linked hyaluronic acid

Abstract

The present invention relates to an LC-MS / MS-based method for quantifying hyaluronic acid, BDDE and BDDE cross-linked hyaluronic acid, and, to a method for identifying and quantifying hyaluronic acid, BDDE and BDDE cross-linked hyaluronic acid, which are contained in a biosample (synovial fluid), by using an LC-MS / MS and multiple heart-cutting (MHC) system.

Description

Quantitative method for hyaluronic acid, BDDE, and BDDE-crosslinked hyaluronic acid based on LC-MS / MS

[0001] The present invention relates to a method for quantitatively analyzing hyaluronic acid, BDDE, and BDDE-crosslinked hyaluronic acid based on LC-MS / MS.

[0002] This work was supported by the Ministry of Science and ICT's Individual Basic Research Project, "Discovering Targets for Immune Anticancer Drug Treatment through Analysis of Multi-Tumor Single-Cell Structural Proteomes" (Project No. 1711194258, 00209456), and the Ministry of Science and ICT's Artificial Intelligence Convergence Innovation Talent Development Project, "Artificial Intelligence Convergence Innovation Talent Development" (Project No. 1711179294, RS-2022-00155857).

[0003] Hyaluronic acid (HA) is a water-soluble polysaccharide, particularly a linear polymer composed of alternating N-acetyl-D-glucosamine and D-glucuronic acid, with a molecular weight of usually 50,000 to 10,000,000 Da or more. It is a major component of the extracellular matrix and is widely distributed in animal tissues.

[0004] It is mainly distributed in the skin of mammals, synovial fluid of joints, vitreous humor of eyes, umbilical cord, serum, cock's comb, etc., and it is also known to exist in the capsule of streptococci. Common methods for obtaining hyaluronic acid include extraction from chicken combs and umbilical cords, and extraction and purification from culturing streptococci such as Lancefield group A and C, and genetically recombinant B. subtilis.

[0005] Hyaluronic acid possesses excellent biocompatibility and does not elicit allergic reactions when implanted into patients. Furthermore, its ability to bind large amounts of water makes it an excellent volumizer for soft tissues. Hyaluronic acid is not species-specific, nor does it exhibit tissue or organ specificity, demonstrating excellent biocompatibility when implanted or injected into living organisms regardless of their origin. This has led to its use in ophthalmic injections and synovial fluid supplements.

[0006] However, since hyaluronidase present in the body hydrolyzes hyaluronic acid, attempts have been made to improve its durability in the body by cross-linking hyaluronic acid with various chemical modifiers or modifying it by attaching functional groups to increase its resistance to hyaluronidase rather than natural hyaluronic acid. Attempts have been made to apply the above methods in combination or to apply excessive amounts of chemical modifiers to further increase the durability in the body, but several unexpected problems have occurred. If a cross-linking agent is applied excessively, excessive denaturation of hyaluronic acid may cause a foreign body reaction or immune rejection reaction in the body. In addition, if 1,4-butanediol diglycidyl ether (BDDE), a widely used bisepoxide-based cross-linking agent, is applied excessively, an imbalance in the ratio of cross-linking agent to hyaluronic acid may form a pendant of incompletely cross-linked cross-linking agent. When such a gel is injected into the body, it may cause an immune rejection reaction or inflammatory reaction due to the pendant.

[0007] Therefore, it is crucial to accurately assess the concentrations of hyaluronic acid and cross-linking agents (e.g., 1,4-Butanediol diglycidyl ether; BDDE) and apply them to clinical (biological) samples. In particular, the simultaneous measurement of hyaluronic acid (HA), BDDE (1,4-Butanediol diglycidyl ether), and BDDE-cross-linked hyaluronic acid concentrations in synovial fluid would enable a more precise understanding of joint disease conditions and biological processes.

[0008] Meanwhile, liquid chromatography-mass spectrometry (LC-MS) is a technology that separates various mixtures based on their physicochemical properties and provides information identifying the type and amount of each separated substance. However, there is currently no standardized method for quantifying hyaluronic acid, BDDE, and cross-linked hyaluronic acid with BDDE using LC-MS analysis. Most analytical methods utilize HPLC (High Performance Liquid Chromatography), which has low sensitivity and specificity, and there is a problem that the analysis conditions are not standardized.

[0009] Korean Patent No. 2527095 discloses a method for measuring the crosslinking rate of a crosslinked hyaluronic acid composition, and Korean Patent No. 2425496 discloses a hyaluronic acid crosslinked product having high elasticity, high viscosity, and high effective crosslinking rate, and a method for producing the same. However, a method for quantitatively determining hyaluronic acid, BDDE, and BDDE crosslinked hyaluronic acid based on LC-MS / MS according to the present invention has not yet been disclosed.

[0010] The present invention was derived from the above-mentioned needs, and the present invention provides a method for quantitatively analyzing hyaluronic acid, BDDE, and BDDE-crosslinked hyaluronic acid based on LC-MS / MS, and confirms that the method can accurately analyze not only the presence or absence of hyaluronic acid and BDDE-crosslinked hyaluronic acid contained in a biological sample (synovial fluid), but also the content thereof, thereby completing the present invention.

[0011] In order to achieve the above purpose, the present invention comprises (1) a step of adding hyaluronic acid hydrolase (Hyaluronidase) to a biological sample that may contain high molecular weight hyaluronic acid or BDDE (1,4-butandiol diglycidyl ether) cross-linked high molecular weight hyaluronic acid at 50 to 2,000 U / ㎖ and performing an enzyme reaction at 30 to 60°C for 1 to 4 days;

[0012] (2) After the above step (1), a step of inactivating the hyaluronic acid hydrolyzing enzyme in a water bath at 90 to 100°C and obtaining the supernatant after centrifugation;

[0013] (3) A step of obtaining mass value 1 (MS1, precursor ion) and mass value 2 (MS2, product ion) represented by m / z (mass to charge) of hyaluronic acid, BDDE (1,4-butandiol diglycidyl ether) or BDDE cross-linked hyaluronic acid contained in the supernatant obtained in step (2) using an LC-MS / MS system; and

[0014] (4) A method for quantitatively analyzing hyaluronic acid, BDDE, and BDDE-crosslinked hyaluronic acid is provided, comprising: a step of calculating the content of hyaluronic acid, BDDE, or BDDE-crosslinked hyaluronic acid by comparing the MS1 and MS2 obtained in the above step (3) with a standard curve obtained from a hyaluronic acid, BDDE, or BDDE-crosslinked hyaluronic acid standard solution.

[0015] The present invention relates to a quantitative method for hyaluronic acid, BDDE and BDDE-crosslinked hyaluronic acid based on LC-MS / MS, and by utilizing a 2D column of MHC (Multiple Heart-Cutting) and MS / MS technique, the content of target components of hyaluronic acid, BDDE and BDDE-crosslinked hyaluronic acid contained in a biological sample (synovial fluid), which is a complex matrix, can be quantitatively analyzed with higher accuracy than before. In particular, it is a quantitative method that can simultaneously obtain the molecular weight of low-molecular-weight HA of 2 to 4 mer and the degree of BDDE crosslinking to HA.

[0016] Figure 1 is a schematic diagram showing a pretreatment method for hyaluronic acid (A) using an enzyme (Hyaluronidase) and hyaluronic acid (B) cross-linked with BDDE.

[0017] Figure 2 is a chromatogram result obtained from LC-MS / MS analysis of standard hyaluronic acid.

[0018] Figure 3 is a chromatogram result obtained from LC-MS / MS analysis of standard BDDE.

[0019] Figure 4 is a chromatogram result obtained from LC-MS / MS analysis of hyaluronic acid cross-linked to standard BDDE.

[0020] Figure 5 is a standard curve for quantification of BDDE.

[0021] Figure 6 is a chromatogram of 4mer hyaluronic acid (4mer-HA) and a standard curve obtained using 50, 100, 500, 1000, 1300, and 2000 ppm of 4mer-HA.

[0022] Figure 7 is a chromatogram of 8mer hyaluronic acid (8mer-HA) and a standard curve obtained using 50, 100, 500, 1000, 1300, and 2000 ppm of 8mer-HA.

[0023] Figure 8 is a chromatogram of 12mer hyaluronic acid (12mer-HA) and a standard curve obtained using 50, 100, 500, 1000, 1300, and 2000 ppm of 12mer-HA.

[0024] Figure 9 is a chromatogram of hyaluronic acid [4mer-HA(M+K)] in a standard solution (1000 ppm HA) and synovial fluid of 7 patients obtained using a 2D LC-MS / MS system equipped with an MHC (Multiple Heart-Cutting) module.

[0025] Figure 10 is a chromatogram of hyaluronic acid [4mer-HA(M+H)] in a standard solution (1000 ppm HA) and synovial fluid of 7 patients obtained using a 2D LC-MS / MS system equipped with an MHC (Multiple Heart-Cutting) module.

[0026] Figure 11 is a chromatogram of hyaluronic acid [8mer-HA(M+H+Na)] in a standard solution (1000 ppm HA) and synovial fluid of 7 patients obtained using a 2D LC-MS / MS system equipped with an MHC (Multiple Heart-Cutting) module.

[0027] Figure 12 is a chromatogram of hyaluronic acid [12mer-HA(M+H+NH4)] in a standard solution (1000 ppm HA) and synovial fluid of 7 patients obtained using a 2D LC-MS / MS system equipped with an MHC (Multiple Heart-Cutting) module.

[0028] Figure 13 is a chromatogram of hyaluronic acid [12mer-HA(M+2H)] in a standard solution (1000 ppm HA) and synovial fluid of 7 patients obtained using a 2D LC-MS / MS system equipped with an MHC (Multiple Heart-Cutting) module.

[0029] Figure 14 is a chromatogram of BDDE-cross-linked hyaluronic acid [4HA-BDDE-2HA(M+)] in a standard solution (Synovian) and synovial fluid of seven patients obtained using a 2D LC-MS / MS system equipped with a Multiple Heart-Cutting (MHC) module.

[0030] Figure 15 is a chromatogram of BDDE-crosslinked hyaluronic acid [2HA-BDDE-2HA(M+)] in a standard solution (Synovian) and synovial fluid of seven patients obtained using a 2D LC-MS / MS system equipped with a Multiple Heart-Cutting (MHC) module.

[0031] Figure 16 is a chromatogram of BDDE cross-linked hyaluronic acid [6HA-BDDE(M+Na)] in a standard solution (Synovian) and synovial fluid of seven patients obtained using a 2D LC-MS / MS system equipped with a Multiple Heart-Cutting (MHC) module.

[0032] Figure 17 is a chromatogram of BDDE cross-linked hyaluronic acid [6HA-BDDE(M+H)] in a standard solution (Synovian) and synovial fluid of seven patients obtained using a 2D LC-MS / MS system equipped with a Multiple Heart-Cutting (MHC) module.

[0033] Figure 18 is a chromatogram of BDDE cross-linked hyaluronic acid [4HA-BDDE(M+)] in a standard solution (Synovian) and synovial fluid of seven patients obtained using a 2D LC-MS / MS system equipped with a Multiple Heart-Cutting (MHC) module.

[0034] Figure 19 is a chromatogram of BDDE-crosslinked hyaluronic acid [2HA-BDDE(M+)] in a standard solution (Synovian) and synovial fluid of seven patients obtained using a 2D LC-MS / MS system equipped with a Multiple Heart-Cutting (MHC) module.

[0035] The present invention comprises (1) a step of adding hyaluronic acid hydrolase (Hyaluronidase) to a biological sample that may contain high molecular weight hyaluronic acid or BDDE (1,4-butandiol diglycidyl ether) cross-linked high molecular weight hyaluronic acid at a concentration of 50 to 2,000 U / ml and performing an enzymatic reaction at 30 to 60°C for 1 to 4 days;

[0036] (2) After the above step (1), a step of inactivating the hyaluronic acid hydrolyzing enzyme in a water bath at 90 to 100°C and obtaining the supernatant after centrifugation;

[0037] (3) A step of obtaining mass value 1 (MS1, precursor ion) and mass value 2 (MS2, product ion) represented by m / z (mass to charge) of hyaluronic acid, BDDE (1,4-butandiol diglycidyl ether) or BDDE cross-linked hyaluronic acid contained in the supernatant obtained in step (2) using an LC-MS / MS system; and

[0038] (4) A method for quantitatively analyzing hyaluronic acid, BDDE and BDDE-crosslinked hyaluronic acid, comprising: a step of comparing MS1 and MS2 obtained in the above step (3) with a standard curve obtained from a hyaluronic acid, BDDE or BDDE-crosslinked hyaluronic acid standard solution to calculate the content of hyaluronic acid, BDDE or BDDE-crosslinked hyaluronic acid;

[0039] The above hyaluronic acid hydrolase (Hyaluronidase) is an enzyme that hydrolyzes hyaluronic acid, hydrolyzing the β-(1,4) glycosidic bond between glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc), which are components of hyaluronic acid, to produce a tetrasaccharide (GlcUA-GlcNAc-GlcUA-GlcNAc), or a mammalian type that decomposes chondroitin sulfate (CS), chondroitin 4-sulfate (C4-S), chondroitin 6-sulfate (C6-S), which are basic components of cartilage and synovial fluid in joints, and dermatan sulfate (DS) present in the mammalian dermis; A leech type that can hydrolyze the β-(1,3) glycosidic bond of hyaluronic acid to produce a tetrasaccharide (GlcNAc-GlcUA-GlcNAc-GlcUA); or a bacterial type that can hydrolyze the β-(1,4) glycosidic bond between glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc); including hyaluronidases.

[0040] The above LC-MS / MS system may include an MHC (multiple heart-Cutting) module, and the MHC is a 2D-LC module that heart-cuts all peaks detected in the first-dimensional separation and then moves them to a second-dimensional device with different selectivity for separation.

[0041] The above LC-MS / MS system is characterized by including a database (DB) including mass values ​​1 (MS1, precursor ion) and mass values ​​2 (MS2, product ion) represented by m / z (mass to charge) of hyaluronic acid (HA), BDDE, and BDDE-crosslinked hyaluronic acid. The database calculates the mass values ​​of hyaluronic acid by size as m / z, which is decomposed through pretreatment and physical energy to be quantified in the system of the present invention, and calculates the mass values ​​in all cases and creates a database. It includes, but is not limited to, the mass-to-charge ratio values ​​(m / z) of MS1 and MS2 of hyaluronic acid (HA), BDDE (1,4-Butanediol diglycidyl ether), and BDDE-crosslinked hyaluronic acid as disclosed in Table 1 below.

[0042] Mass-to-charge ratio values ​​(m / z) of MS1 and MS2 of standard hyaluronic acid (HA), 1,4-butanediol diglycidyl ether (BDDE), and BDDE-crosslinked hyaluronic acid Precursor NamePrecursor FormulaPrecursor Adductprecursor m / z(MS 1)Precursor ChargeProduct m / z(MS 2)Product Charge2HA-BDDEC 24 H 41 N1O 16 M+599.2419861599.20054914HA-BDDEC 38 H 62 N2O 27 M+978.3534471978.30054914HA-BDDEC 38 H 62 N2O 27 M+978.3534471599.20054916HA-BDDEC 52 H 83 N3O 38 M+H1358.47273211358.40054916HA-BDDEC 52 H 83 N3O 38M+H1358.4727321978.30054916HA-BDDEC 52 H 83 N3O 38 M+H1358.4727321599.20054916HA-BDDEC 52 H 83 N3O 38 M+Na1380.45467711380.40054916HA-BDDEC 52 H 83 N3O 38 M+Na1380.4546771978.30054916HA-BDDEC 52 H 83 N3O 38 M+Na1380.4546771599.20054912HA-BDDE-2HAC 38 H 60 N2O 26 M+960.3428821960.30054914HA-BDDE-2HAC 52 H 81 N3O 37 M+1339.45434311339.10054914HA-BDDE-2HAC 52 H 81 N3O 37 M+1339.4543431687.60054914HA-BDDE-2HAC 52 H 81 N3O 37 M+1339.4543431669.10054914HA-BDDE-2HAC 52 H 81 N3O 37 M+1339.4543431458.40054914HA-BDDE-2HAC 52 H 81 N3O 37 M+1339.4543431446.200549112HAC 84 H 126 N6O 66 [M+H+NH4]1146.85493321147.500549212HAC 84 H 126 N6O 66 [M+H+NH4]1146.8549332931.200549212HAC84 H 126 N6O 66 [M+H+NH4]1146.8549332797.200549212HAC 84 H 126 N6O 66 [M+H+NH4]1146.8549332785.900549212HAC 84 H 126 N6O 66 [M+H+NH4]1146.8549332770.800549212HAC 84 H 126 N6O 66 [M+H+NH4]1146.8549332767.500549212HAC 84 H 126 N6O 66 [M+H+NH4]1146.8549332759.300549212HAC 84 H 126 N6O 66 [M+H+NH4]1146.8549332248.500549212HAC 84 H 126 N6O 66 [M+H+NH4]1146.8549332175.000549212HAC 84 H 126 N6O 66 [M+2HA]1138.34165821138.400549212HAC 84 H 126 N6O 66 [M+2HA]1138.3416582931.200549212HAC 84 H 126 N6O 66 [M+2HA]1138.3416582797.200549212HAC 84 H 126 N6O 66 [M+2HA]1138.3416582785.900549212HAC 84 H 126 N6O 66 [M+2HA]1138.3416582770.800549212HAC 84 H 126 N6O 66[M+2HA]1138.3416582767.500549212HAC 84 H 126 N6O 66 [M+2HA]1138.3416582759.300549212HAC 84 H 126 N6O 66 [M+2HA]1138.3416582248.500549212HAC 84 H 126 N6O 66 [M+2HA]1138.3416582175.000549210HAC 70 H 105 N5O 55 [M+2HA]948.7859282931.200549210HAC 70 H 105 N5O 55 [M+2HA]948.7859282797.200549210HAC 70 H 105 N5O 55 [M+2HA]948.7859282785.900549210HAC 70 H 105 N5O 55 [M+2HA]948.7859282770.800549210HAC 70 H 105 N5O 55 [M+2HA]948.7859282767.500549210HAC 70 H 105 N5O 55 [M+2HA]948.7859282759.300549210HAC 70 H 105 N5O 55 [M+2HA]948.7859282248.500549210HAC 70 H 105 N5O 55 [M+2HA]948.7859282175.00054928HAC 56 H 84 N4O 44 [M+H+Na]770.2211702797.20054928HAC 56 H 84 N4O44 [M+H+Na]770.2211702785.90054928HAC 56 H 84 N4O 44 [M+H+Na]770.2211702770.80054928HAC 56 H 84 N4O 44 [M+H+Na]770.2211702767.50054928HAC 56 H 84 N4O 44 [M+H+Na]770.2211702759.30054928HAC 56 H 84 N4O 44 [M+H+Na]770.2211702248.50054928HAC 56 H 84 N4O 44 [M+H+Na]770.2211702175.00054924HAC 28 H 42 N2O 22 [M+H]759.2223731759.30054914HAC 28 H 42 N2O 22 [M+H]759.2223731175.00054914HAC 28 H 42 N2O 22 [M+K]797.1860791797.10054914HAC 28 H 42 N2O 22 [M+K]797.1860791785.90054914HAC 28 H 42 N2O 22 [M+K]797.1860791175.0005491BDDEC 10 H 18 O4[M+H]203.1277851147.0005491BDDEC 10 H 18 O4[M+H]203.1277851129.1005491BDDEC 10 H 18 O4[M+H]203.127785173.1005491BDDEC 10 H18 O4[M+H]203.127785157.3005491BDDEC 10 H 18 O4[M+H]203.127785143.3005491BDDEC 10 H 18 O4[M+H]203.127785129.3005491

[0043] The substances analyzed in Table 1 above are BDDE cross-linked hyaluronic acid (HA); hyaluronic acid (HA); and BDDE (1,4-butanediol diglycidyl ether).

[0044] The above biological sample is preferably synovial fluid, but is not limited thereto, and any biological sample containing hyaluronic acid may be used.

[0045] The hyaluronic acid contained in the above biological sample is preferably a high molecular weight polymer of 5 to 20,000 kDa, more preferably 500 to 2500 kDa, but is not limited thereto.

[0046]

[0047] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0048]

[0049] Example 1. Pretreatment for quantitative analysis of hyaluronic acid (HA), 1,4-butanediol diglycidyl ether (BDDE), and hyaluronic acid cross-linked with BDDE based on LC-MS / MS.

[0050] In order to analyze the content of hyaluronic acid (HA), BDDE (1,4-Butanediol diglycidyl ether), and BDDE-crosslinked hyaluronic acid based on LC-MS / MS, high-molecular-weight HA and BDDE-crosslinked hyaluronic acid were hydrolyzed with an enzyme (Hyaluronidase) to reduce the molecular weight (Fig. 1).

[0051] Specifically, (1) After treating hyaluronic acid (HA) and BDDE-crosslinked hyaluronic acid with an enzyme (Hyaluronidase) at a concentration of 50 to 2,000 U / ㎖, an enzyme reaction was performed at 50°C for 3 days.

[0052] (2) When the reaction was completed, the enzyme (Hyaluronidase) was inactivated in a 90°C water bath, and the supernatant was collected after centrifugation and used for LC-MS / MS analysis.

[0053]

[0054] Example 2. Analysis conditions for hyaluronic acid (HA), BDDE (1,4-Butanediol diglycidyl ether), and BDDE-crosslinked hyaluronic acid based on LC-MS / MS system

[0055] Each hyaluronic acid (HA), BDDE (1,4-Butanediol diglycidyl ether), and hyaluronic acid cross-linked with BDDE, which were decomposed into a form that could be analyzed by LC-MS / MS through the pretreatment performed in Example 1, were used as samples for LC-MS / MS analysis. The LC-MS / MS analysis method was performed using the mass value (MS1, precursor ion) having a specific m / z (mass to charge) of the target substance and the mass value (MS2, product ion) of the fragment having a characteristic m / z generated when the target substance is destroyed (collision). The analysis conditions are disclosed in Table 2 below.

[0056] The precursor ion / product ion obtained from MS1 / MS2, respectively, are named as specific mass fingerprints (transitions) for each target substance and are expressed as mass pairs, such as "978.353 / 599.200 m / z." The m / z of hyaluronic acid, BDDE, and BDDE-crosslinked hyaluronic acid obtained using standard solutions are shown in Table 1.

[0057] LC-MS Conditions Column ZORBAX RRHD Eclipse Plus C18, 95Å, 2.1×150mm, 1.8μm, 1200 bar pressure limit Column temperature 40℃ Mobile phase A: Water containing 0.1% formic acid B: Acetonitrile (ACN) containing 0.1% formic acid Gradient 0 min, 98% A 4 min, 98% A 6 min, 94% A 25 min, 75% A 26.5 min, 40% A 27.50 min, 40% A 27.51 min, 98% A 30 min, 98% A Flow rate 0.4㎖ / min Injection amount 5㎕ Ion mode Positive ion mode (ESI+) Gas temperature 150℃ Gas flow 12ℓ / min Nebulizer 30psi Sheath Gas temperature 250℃ Sheath Gas flow12ℓ / minCapillary2500V

[0058] As a result, as disclosed in Figures 2 to 4, the retention time and peak area for each peak were confirmed from the chromatograms of hyaluronic acid, BDDE, and BDDE-crosslinked hyaluronic acid in the standard solution.

[0059]

[0060] Example 3. Quantitative curves of hyaluronic acid (HA), 1,4-butanediol diglycidyl ether (BDDE), and hyaluronic acid cross-linked with BDDE.

[0061] The peak area value of each substance generated at each concentration was calculated through LC-MS / MS analysis (Figs. 2-4), and then the standard curve and quantitative formula for hyaluronic acid and BDDE were derived (Figs. 5-8).

[0062]

[0063] Example 4. Quantitative analysis of hyaluronic acid (HA) and BDDE-crosslinked hyaluronic acid contained in joint fluid collected from 7 patients.

[0064] Synovial fluid was collected from seven patients suffering from arthritis. To determine the content of hyaluronic acid and BDDE-crosslinked hyaluronic acid in the synovial fluid according to the method of the present invention, the synovial fluid was pretreated with a hyaluronic acid hydrolase, centrifuged, and the supernatant was collected. Subsequently, the supernatant was subjected to quantitative analysis of hyaluronic acid (HA) and BDDE-crosslinked hyaluronic acid in a complex matrix (synovial fluid) using an LC-MS / MS system equipped with a Multiple Heart-Cutting (MHC) module. The MHC module enhanced the separation of components in the complex sample, providing excellent separation performance capable of identifying target substances in the complex sample.

[0065] As a result, LC chromatograms as disclosed in Figures 9 to 19 were obtained, and some area values ​​from the chromatograms were adopted and substituted into each standard curve established from the standard solution to confirm the content of hyaluronic acid in the synovial fluid according to the cut form as disclosed in Table 3 below.

[0066] Patient number 4mer-HA(M+K), ㎍ / ㎖ 4mer-HA(M+H), ㎍ / ㎖ 8mer-HA(M+H+Na), ㎍ / ㎖ 12mer-HA(M+2H), ㎍ / ㎖ 12mer-HA(M+H+NH4), ㎍ / ㎖ 2 21356.5 1808.7 2004.91482.9959.3 241435.41745.3 1806.6 1632.2820.3 361589.41788.42163.01640.21011.8371716.81847.32193.41591.2961.6542303.02043.82084.21837.7993.0591743.81848.72222.21751.5877.6851295.51369.41027.11256.6349.9

Claims

1. (1) A step of adding hyaluronic acid hydrolase (Hyaluronidase) to a biological sample that may contain high molecular weight hyaluronic acid or BDDE (1,4-butandiol diglycidyl ether) cross-linked high molecular weight hyaluronic acid at a concentration of 50 to 2,000 U / ㎖ and performing an enzyme reaction at 30 to 60°C for 1 to 4 days; (2) After the above step (1), a step of inactivating the hyaluronic acid hydrolyzing enzyme in a water bath at 90 to 100°C and obtaining the supernatant after centrifugation; (3) A step of obtaining mass value 1 (MS1, precursor ion) and mass value 2 (MS2, product ion) represented by m / z (mass to charge) of hyaluronic acid, BDDE (1,4-butandiol diglycidyl ether) or BDDE cross-linked hyaluronic acid contained in the supernatant obtained in step (2) using an LC-MS / MS system; and (4) A method for quantitatively analyzing hyaluronic acid, BDDE, and BDDE-crosslinked hyaluronic acid, comprising: a step of comparing MS1 and MS2 obtained in the above step (3) with a standard curve obtained from a hyaluronic acid, BDDE, or BDDE-crosslinked hyaluronic acid standard solution to calculate the content of hyaluronic acid, BDDE, or BDDE-crosslinked hyaluronic acid; 2. A quantitative method according to claim 1, characterized in that the LC-MS / MS system includes a multiple heart-Cutting (MHC) module.

3. A quantitative method according to claim 1, wherein the LC-MS / MS system comprises a database (DB) including mass value 1 (MS1, precursor ion) and mass value 2 (MS2, product ion) represented by m / z (mass to charge) of hyaluronic acid (HA), BDDE, and BDDE cross-linked hyaluronic acid.

4. A quantitative method according to claim 1, characterized in that the biological sample is synovial fluid.

5. A quantitative method according to claim 1, characterized in that the hyaluronic acid contained in the biological sample is a high molecular weight polymer of 5 to 20,000 kDa.

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