Corresponding author: Nataliia Hudz ( natali_gudz@ukr.net ) Academic editor: Plamen Peikov
© 2019 Nataliia Hudz, Dmytro Leontiev, Piotr P. Wieczorek.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hudz N, Leontiev D, Wieczorek PP (2019) Spectral characteristics of 5hydroxymethylfurfural as a related substance in medicinal products containing glucose. Pharmacia 66(3): 121125. https://doi.org/10.3897/pharmacia.66.e35969

Objectives: To study 5hydroxymethylfurfural (5HMF) spectral characteristics aiming at their future application in analytical procedures and their validation for the determination of 5HMF in liquid products containing glucose after sterilization. Method: Direct spectrophotometric method for the determination of 5HMF using the molar absorption coefficient at the absorption maximum (284 nm).
Results and discussion: aqueous 5HMF solutions have strong absorption in the ultraviolet range below 310 nm and give two absorption maxima at wavelengths of 229–230 nm and 284 nm. An excellent linear relationship between absorbance and 5HMF concentration was observed in the concentration range of 2.0–10.0 mg/l. The linear dependence passes through the origin. The molar absorption coefficients of 5HMF were determined and found to be 3007 mol^{−1}•L•cm^{−1}at 229–230 nm and 16070 mol^{−1}•L•cm^{−1}at 284 nm. The use of the molar absorption coefficient of 5HMF stated in the Pharmacopeia of the United States of America for determining 5HMF in polydextrose (16830 mol^{−1}·L·cm^{−1}at 283 nm) gives recovery results for model solutions of reference substance of 5HMF that are acceptable from the point of view of the requirements of the State Pharmacopeia of Ukraine for methods of quantitative determination of impurities. However, other values of the molar absorption coefficient (17000 and 22700 mol^{−1}•L•cm^{−1}) given in the scientific publications are unsuitable for the quantitative determination of 5HMF as an impurity in medicinal products.
Conclusion: The molar absorption coefficient (16830 mol^{−1}•L•cm^{−1}at 284 nm) may be used to quantify 5HMF as an impurity in medicinal products containing glucose. For a specific medicinal product, a full validation of the analytical procedure of the 5HMF determination is required taking into account the composition of this product.
5hydroxymethylfurfural, spectrophotometric method, molar absorption coefficient, medicinal products containing glucose
In the pharmaceutical industry, heat sterilization is widely applied to medicinal products in liquid dosage forms. Steam sterilization is considered to be sterilization of choice (
Quality control of peritoneal dialysis solutions is performed according to the monograph of the British Pharmacopeia. This monograph provides analytical procedures for measuring the 5HMF concentrations based on the colored product which is formed as a result of the reaction between 5HMF, ptoluidine in 2propanol containing glacial acetic acid and barbituric acid (
The State Pharmacopoeia of Ukraine establishes requirements for the maximum permissible uncertainty of analysis results (max∆_{As}) based on the risk of making an incorrect conclusion on compliance with specifications (The State Pharmacopoeia of Ukraine). A reliability level of 95% is considered as acceptable. It is recommended that max∆_{As} should not exceed 5% for quantitative determination of impurities. The existing methods for determining 5HMF content were not analyzed in terms of the risk of making an incorrect decision and in terms of the requirements for max∆_{As}.
The primary aim of this research was to study the 5HMF spectral characteristics, linearity, intermediate precision, range, detection limit, quantitation limit of the analytical procedure of the 5HMF determination and estimate the possibility of using molar absorption coefficient ԑ for 5HMF assay in liquid medicinal products containing glucose from the point of view of the risk of making an incorrect decision in accordance with the approaches of the State Pharmacopeia of Ukraine (The State Pharmacopoeia of Ukraine). Our secondary goal was to study the repeatability of the 5HMF spectral characteristics and compare them with literature data as well.
5HMF (analytical standard, batch number: BCBR3219V) was purchased from SigmaAldrich (USA). It was dissolved in purified water before analysis. As stock solutions were ones with 5HMF concentrations of 19.7 mg/L (the first experiment) and 19.9 mg/L (the second experiment for the intermediate precision estimation).
Direct spectrophotometric method of the 5HMF determination was employed.
The five aqueous 5HMF solutions with the concentrations in the range of 2–10 mg/L were prepared using purified water as a solvent. The 5HMF concentrations were calculated using the known values of the molar absorption coefficient ԑ for 5HMF at the wavelength of maximum absorption at 283284 nm according to the following formula:
$C=\genfrac{}{}{0.1ex}{}{A\u2022M\cdot m\cdot 1{0}^{4}}{10\u2022\epsilon}=\genfrac{}{}{0.1ex}{}{A\u2022M\cdot m\cdot \u20221{0}^{3}}{\epsilon}$
where C – 5HMF concentration in mg/L, A – solutions absorbance at an absorption maximum of 283–284 nm, M.m. – molar mass of 5HMF (126 g/mol), ԑ – molar absorption coefficient.
Spectrophotometer Hitachi U2810 (Hitachi HighTechnologies Corporation, Japan) was used. A 1cm quartz cell was used over the range of 200 to 350 nm.
It was shown in a published paper (
Moreover, the ratio of the absorbances at these two absorbtion maxima (A_{284}: A_{228}) was in the range of 5.04–5.65 that almost conforms to data provided by
For the study of intermediate precision experiments were performed twice. The second experiment was repeated in 5 months using the same spectrophometer and the same batch of 5HMF as a reference substance. Fig.
The detailed spectral characteristic of 5HMF in the first and second experiments are given in Tables
Detailed spectral characteristic of 5HMF (01 August 2016).
5HMF concentration, mg/L  Wavelengths of maximum absorption, λ_{max}, nm  Absorbance at the absorption maxima (A)  Ratio A_{λ1} : A_{λ2}  Found values of ԑ at  % recovery at ԑ values  

229 nm  284 nm  16830  17000  22700  
1.97  λ_{1} = 284.2  0.252  5.04  3198  16118  95.77  94.81  71.00 
λ_{2} = 228.0  0.050  
3.94  λ_{1} = 283.8  0.504  5.54  2910  16118  95.77  94.81  71.00 
λ_{2} = 229.4  0.091  
5.91  λ_{1} = 283.8  0.751  5.65  2836  16011  95.13  94.18  70.53 
λ_{2} = 229.4  0.133  
7.88  λ_{1} = 284.0  0.991  5.54  2862  15846  94.15  93.21  69.81 
λ_{2} = 229.0  0.179  
9.85  λ_{1} = 284.0  1.237  5.50  2878  15824  94.02  93.08  69.71 
λ_{2} = 229.2  0.225  
mean ± SD  λ_{1} = 284.0 ± 0.2  –  5.45 ± 0.24  2937 ± 149  15983 ± 143  94.97 ± 0.85  94.02 ± 0.84  70.41 ± 0.62 
λ_{2} = 229.0 ± 0.6  
mean ± % RSD  –  –  –  2937 ± 5.1%  15983 ± 0.89%  94.97 ± 0.90%  94.02 ± 0.89%  70.41 ± 0.88% 
Detailed spectral characteristic of 5HMF (10 December 2016).
5HMF concentration, mg/L  Wavelengths of maximum absorption, λ_{max}, nm  Absorbance at the absorption maxima (A)  Ratio A_{λ1}: A_{λ2}  Found values of ԑ at  % recovery at ԑ values  

229 nm  284 nm  16830  17000  22700  
1.99  λ_{1}=284.0  0.258  5.27  3103  16336  97.06  96.09  71.96 
λ_{2} = 230.2  0.049  
3.98  λ_{1} = 284.0  0.507  5.28  3039  16051  95.37  94.42  70.71 
λ_{2} = 229.5  0.096  
5.97  λ_{1} = 284.0  0.774  5.27  3103  16336  97.06  96.09  71.96 
λ_{2} = 229.5  0.147  
7.96  λ_{1} = 284.0  1.014  5.34  3008  16051  95.37  94.42  70.71 
λ_{2} = 229.5  0.190  
9.95  λ_{1} = 284.0  1.264  5.31  3027  16006  95.11  94.16  70.51 
λ_{2} = 229.5  0.238  
mean ± SD  λ_{1} = 284.0 ± 0.0  –  5.29 ± 0.03  3076 ± 56  16156 ± 165  95.99 ± 0.98  95.04 ± 0.97  71.17 ± 0.73 
λ_{2} = 229.7 ± 0.3  
mean ± % RSD  –  –  –  3076 ± 1.82%  16156 ± 1.02%  95.99 ± 1.02%  95.04 ± 1.02%  71.17 ± 1.03% 
The condition for the correct application of the molar absorption coefficient ԑ is also the passage of a linear dependence through the origin. To calculate the confidence interval, onetailed Student coefficient for the level of reliability of 95% and number of freedom degrees n = 5 – 2 = 3 (t = 2.3534) was used. For the both experiments, the yintercepts did not exceed their confidence interval that confirms the correctness of using the molar absorption coefficient to determine 5HMF content.
We also experimentally determined molar absorption coefficient for 5HMF at 229–230 nm. To the best of our knowledge, only one available publication provides the value of the molar absorption coefficient for 5HMF at 228 nm, which is equal to 3000 L•mol^{1}•cm^{1}(
$\epsilon =\genfrac{}{}{0.1ex}{}{A\u2022M\cdot m\cdot \u2022{0}^{3}}{C}$ ,
where A – solutions absorbance at the first absorption maximum, M.m. – molar mass of 5HMF (126 g/mol), C – 5HMF concentration in mg/L.
The molar absorption coefficient of 5HMF at 229–230 nm was determined and found to be 3007 mol^{−1}•L•cm^{−1} that is in line with data of
It can be seen that very similar values were obtained for the molar absorption coeffitients of 5HMF in the same laboratory at different days. The mean of two experiments was 3007 mol^{−1}•L•cm^{−1} ± 3.26% at 229–230 nm and 16070 mol^{−1}•L•cm^{−1} ± 0.76% at 284 nm. These deviations (3.26% and 0.76%) did not exceed the requirements of the State Pharmacopeia of Ukraine for max∆_{As} for methods of quantitative determination of impurities (max∆_{As} ≤ 5%). Consequently, the elaborated procedure is characterized by good intermediate precision.
The following values of the molar absorption coefficient ԑ for 5HMF at an absorption maximum of 283–284 nm were used for the estimation of the analytical procedure recovery:
The average recovery values for the molar absorption coefficients were calculated for each experiment (Tables
Limit of detection (LoD) and limit of quantification (LoQ) were calculated from the SD of yintercept and the slope of the calibration lines (
Thus, the developed direct spectrophotometric method of the 5HMF quantitative determination using the molar absorption coefficient at 283–284 nm is potentially suitable for medicinal products containing glucose.
A linear relationship between absorbance and HMF concentration was observed in the concentration range of 2–10 mg/l. The linear dependence passes through the origin. The molar absorption coefficients of 5HMF were determined and found to be 3007 mol^{−1}·L·cm^{−1}at 229–230 nm and 16070 mol^{−1}•L•cm^{−1}at 284 nm. The use of the molar absorption coefficient of 5HMF at 284 nm gives results that are acceptable from the point of view of the requirements of the State Pharmacopeia of Ukraine for methods of quantitative determination of impurities. The method could be potentially applied to the determination of 5HMF, provided that validation of the analytical procedure for each medicinal product is studied.
Coauthor Natalia Hudz is grateful to the International Visegrad Fund for providing scholarship for studies related to solutions for dialysis therapy.