Measurement of Low-Molecular-Mass Heparins based on the Requirements of the European Pharmacopoeia

Download PDF August 11, 2022

Introduction

LC-4000 Series HPLC System
LC-4000 Series HPLC System

Heparins are a mucopolysaccharide obtained from porcine small intestine and is widely used in the pharmaceutical field as an  anticlotting drug for treatment and prevention of thromboembolism, treatment of disseminated intravascular coagulation syndrome, and as an anti-blood clotting agent for using extracorporeal blood circulating apparatus such as blood dialysis and artificial heart/lung.

Low-molecular-mass heparin is produced by enzymatic treatment and chemical treatment of unfractionated heparin. Low-molecular-mass heparin is classified as parnaparin, dalteparin, enoxaparin, and so on by a decomposition method and molecular weight distribution.

The European Pharmacopoeia 8th Edition (EP), molecular weight measurement method is defined in a pharmaceutical products article for low-molecular-mass heparin and the average molecular weight and molecular weight distribution are also defined in the pharmaceutical article for low-molecular-mass heparin (sodium) and low-molecular-mass heparin (calcium). SEC with a UV-Vis detector and Refractive Index detector are the defined measurement method. Using this method, a molecular weight calibration curve using low-molecular-mass heparin is created by using low-molecular-mass heparin as a reference sample. In common molecular weight measurement methods using SEC, a calibration curve is created from the retention volume and molecular weight. This common method uses oligomers and polymers with known molecular weight as the standards. However, molecular weights have to be calculated by standardizing coefficient and intensity ratio of RI and UV to create the calibration curve. This analysis is typically too complicated for normal commercial  SEC software to be used.

In this application note, molecular weight measurement is made based on the European Pharmacopoeia method for the analysis of average molecular weight and molecular weight distribution using the ChromNAV Low-Molecular-Mass Heparin Molecular Weight Calculation program for ChromNAV.

Experimental

Equipment
PumpPU-4185
Pump OptionDegasser
AutosamplerAS-4050
Autosampler OptionTC Unit
Column OvenCO-4060
DetectorUV-4075, RI-4030

Conditions
ColumnTSKgel G2000SWXL (7.8 mm I.D. x 300 mm L, 5 µm)
EluentAqueous solution of 0.2 M anhydrous sodium sulfate (adjusted to pH 5.0 with 0.05 M sulfuric acid)
Flow Rate0.5 ML/MIN
Column Temp30°C
Wavelength234 mL
Injection Volume25 mL
Standard20 mg of low-molecular-mass heparin for calibration CRS*
in 2 mL of the eluent
*number average relative molecular mass: 3,800
Sample20 mg of Parnaparin sodium CRS in 2 mL of the eluent
20 mg of Dalteparin sodium CRS in 2 mL of the eluent

APP Note 747014H structure image

Keywords

Low-molecular-mass heparin, Parnaparin sodium, Dalteparin sodium, European Pharmacopoeia, Molecular weight measurement, UV detector, Refractive Index detector, Low-Molecular-Mass Heparin Molecular weight calculation program

Results

APP note 747014 fig 1 Chromatogram of low-molecular-mass heparin for molecular weight measurement.
Figure 1. Chromatogram of low-molecular-mass heparin for molecular weight measurement. Figs. 2 and 3 chromatograms for parnaparin and dalteparin.
APP Note 747014H Figure 2. Chromatograms of parnaparin
Figure 2. Chromatograms of parnaparin
APP Note 747014H Figure 3. Chromatograms of dalteparin
Figure 3. Chromatograms of dalteparin

The calibration curve is created using a chromatogram of low-molecular-mass heparin for molecular weight measurement [Create Calibration Curve] displayed in the [Heparin Molecular Weight Calculation Method] window. In figure 4, the calibration curve is created by using three replicate measurements of low-molecular-mass heparin for molecular weight measurement. In ChromNAV the calibration curve can be created automatically.

APP Note 747014H fig 4
Figure 4. ChromNAV [Heparin Molecular Weight Calculation Method ] window- [Create Calibration Curve] display
(A) chromatogram, (B) calibration curve, (C) Information about calibration curve, (D) Parameters of molecular weight calibration curve,
(E) Chromatogram list

The [Molecular Weight Calculation Parameter] displayed in [Heparin Molecular Weight Calculation Method] window is used for setting the range for calculation of molecular weight against a sample. Figure 5 shows the [Molecular Weight Calculation Parameter] display of parnaparin after setting the range which is refined in the European Pharmacopoeia. The range for calculating molecular weight distribution depends on the heparin to be measured, a calibration is created for low-molecular-mass heparin (sodium), and another for dalteparin (sodium).

APP Note 747014H fig 5 ChromNAV [Heparin Molecular Weight Calculation Method] window - [Molecular Weight Calculation Parameter] display<br /> (A) chromatogram, (B) Calculation mode and method, (C) Range for molecular weight calculation,<br /> (D) Percentage for molecular weight range
Figure 5. ChromNAV [Heparin Molecular Weight Calculation Method] window – [Molecular Weight Calculation Parameter] display
(A) chromatogram, (B) Calculation mode and method, (C) Range for molecular weight calculation,
(D) Percentage for molecular weight range

The calculation results is displayed in [Heparin Molecular Weight Calculation Results View] by applying the prepared [Heparin Molecular Weight Calculation Method] to a sample. Figure 6 shows [Heparin Molecular Weight Calculation Results View] for parnaparin (sodium) and dalteparin (sodium). These are created by applying prepared the [Heparin Molecular Weight Calculation Method] to their RI chromatograms. In this view, the UV chromatogram and the calibration curve can be overlaid with the RI chromatogram of the sample for determining the calculated average molecular weight and molecular weight distribution according to the European Pharmacopoeia, the calculation result table shown in figure 6 (c) is useful. In this application both the parnaparin (sodium) and dalteparin (sodium) satisfy the levels in the European Pharmacopoeia (Table 1).

APP Note 747014H fig 6
Figure 6. ChromNAV [Heparin Molecular Weight Calculation Result View] display
(A) Chromatogram, (B) tool buttons, (C) Heparin Molecular Weight Calculation Results Table

Table 1. Average molecular weight and molecular weight distribution in low-molecular- mass heparin sodium and calcium article of European Pharmacopoeia

Low-Molecular-Mass HerapinsRange of Average
Molecular Weight
Molecular Weight Distribution
M < 2000M < 3000M 2000 - 4000M 2000 - 8000M 3000 - 8000M > 8000
Parnaparin Sodium4000 - 6000≤ 30%50 - 60%
Dalteparin Sodium5600 - 6400≤ 13%15 - 25%
Enoxaparin Sodium3800 - 500012 - 20%68 - 82%
Tinzaparin Sodium5500 - 7500≤ 10%60 - 72%22 - 36%
Nadroparin Sodium3600 - 5000≤ 15%35 - 55%75 - 95%

References

  1. Acupuncture, Thrombus, Hemostatic, 19 (2), 187-190 (2008).
  2. European Pharmacopoeia 8.0 volume II,“Heparins, low-molecular-mass monograph 0828”, (2014), Council of Europe.
  3. European Pharmacopoeia 8.0 volume II,“Parnaparin sodium monograph 1252”, (2014), Council of Europe.
  4. European Pharmacopoeia 8.0 volume II,“Parnaparin sodium monograph 1252”, (2014), Council of Europe.
  5. European Pharmacopoeia 8.0 volume II,“Dalteparin sodium monograph 1195”, (2014), Council of Europe.
  6. European Pharmacopoeia 8.1 supplement,“Enoxaparin sodium monograph 1097”, (2014), Council of Europe.
  7. European Pharmacopoeia 8.0 volume II,“Tinzaparin sodium monograph 1271”, (2014), Council of Europe.
  8. European Pharmacopoeia 8.0 volume II,“Nadroparin calcium monograph 1134”, (2014), Council of Europe.

CodeModelDescription
WWA3PU-4185Semi-Micro Pump
C477DG-4000-044-Channel Analytical Degasser Unit
RR95AS-4050Autosampler
C502TC-4000-1Sample Temperature Control Unit
RRA1CO-4060Column Oven
WW88UV-4075UV/Vis Detector
WW94RI-4030Refractive Index Detector
R354RI-4030Bottle Stand
TSK gel G2000SWxl (7.8 mml.D. x 300 mmL, 5 μm)
C006 x 4RI-4030LC-Net CG cable x 4
ChromNAV V2ChromNAV V2 + PC
C294Low-Molecular-Mass Heparins Calculation Program
078CHPLC Start Up Kit for LC-4000
089CMaintenance Tool Kit

In this analysis, please use the pump cleaning mechanism due to a high salt concentration mobile phase.

Fresh ultrapure water is required for the cleaning mechanism before each measurement.

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About the Author

DJ Tognarelli received his B.S. from the University of Richmond where he studied Analytical Chemistry. He is a Chromatography Applications Scientist at JASCO.