Determination of the anticoagulant activity of low molecular weight heparins by micellar electrokinetic chromatography combined with on-column enzymatic reaction

doi:10.3724/SP.J.1123.2020.07010

Abstract

Abstract:

Low molecular weight heparins (LMWHs) have largely replaced heparin for the treatment and prevention of thrombosis because of their various advantages over unfractionated heparins (UFHs) such as less bleeding, greater bioavailability, and more predictable anticoagulant effects. For special groups of patients, such as pregnant women, children, and patients with renal failure, it is necessary to monitor the anticoagulant activity of LMWHs in the blood. The traditional method used to determine the anticoagulant activity of heparin is the coagulation test. However, the results are various from different laboratories and different reagents. In contrast, the chromogenic substrate method is more accurate, sensitive and is easy to automate. Here, a method for the determination of the anticoagulant activity of LMWHs was developed by using a capillary-electrophoresis-based substrate chromogenic method.

In this method, micellar electrokinetic chromatography (MEKC) was used in combination with electrophoretically mediated microanalysis to determine the anti-factor Xa (FXa) activity of LMWHs. The inhibition was measured by employing a chromogenic peptide substrate (CPS) with a p -nitroaniline (p -NA) moiety as the chromophore. The injection end of the capillary was used as a microreactor in which solutions of LMWHs, antithrombin Ⅲ (ATⅢ), FXa and CPS were successively injected and mixed by using diffusion, the transverse diffusion of laminar flow profiles and applied voltage. The reaction product p -NA was separated from unreacted CPS and sample matrix by using the MEKC mode with discontinuous background electrolyte system. The produced p -NA was baseline separated from the other components and detected at 380 nm to obtain maximum sensitivity. The amount of p -NA was inversely proportional to the activity of LMWHs in the sample. To improve the accuracy of quantification and the method repeatability of methods, nitrofurantoin (NF) was selected as the internal standard, which was added to the solution of CPS. The method was validated and used to measure a set of samples. The method is characterized by automation, good repeatability, high sensitivity, and cost-effectiveness. Additionally, the method does not interfere by the sample matrix, and thus can be used to monitor the anticoagulant activity of LMWHs in plasma.

Key words: micellar electrokinetic chromatographic (MEKC), on-column enzymatic reaction, low molecular weight heparins (LMWHs), anticoagulant

ZHANG Mingyu, KANG Jingwu. Determination of the anticoagulant activity of low molecular weight heparins by micellar electrokinetic chromatography combined with on-column enzymatic reaction[J]. Chinese Journal of Chromatography, 2020, 38(10): 1243-1248.

Figures/Tables 7

Fig. 1 Schematic presentation for mixing reactants inside the capillary by transverse diffusion of laminar flow a. two reactants are injected; b. apply a short-time injection with high pressure; c. let stand to mix the two reactants completely.

Fig. 2 Effect of the mixing time of anti-factor Xa (FXa)and chromogenic peptide substrate (CPS) on the reaction yields of p -nitroaniline (p -NA)

Fig. 3 Standard curve of p -NA (n =3) Concentrations of p -NA: 0.025, 0.05, 0.1, 0.25, 0.5, 1.0 mmol/L; concentration of nitrofurantoin (NF): 0.1 mmol/L; Injection: 140 Pa×10 s. y : peak area ratios of p -NA to NF; x : concentration, mmol/L; R 2 : correlation coefficient.

Fig. 4 Electropherograms for the determination of anti-FXa activity of enoxaparin sodium (E) Contents of E: S1 , 0.087; S2 , 0.111; S3 , 0.143; S4 , 0.183 IU/mL.

Table 1 Intra-day and inter-day RSDs of migration times, peak areas, and peak area ratios of p -NP 1.61

Item	Intra-day RSD (n =10)/%	Inter-day RSD (n =3)/%
Migration time	0.13	0.55
Peak area	4.22	12.8
Peak area ratio	2.69	1.61

Fig. 5 Calibration curve of E constructed by peak area ratio vs log concentration value

Table 2 Potency results of two batches of samples

Sample	Assumed potency/(IU/mL)	Determined potency/(IU/mL)	S_M / %	FL/ %
S_M : standard error; FL: fiducial limit.
E-1	10000	12028	0.90	4.9
E-2	12000	13282	0.73	4.0

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