Corresponding author: Nataliia Hudz (

^{−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 5-HMF stated in the Pharmacopeia of the United States of America for determining 5-HMF in polydextrose (16830 mol^{−1}·L·cm^{−1}at 283 nm) gives recovery results for model solutions of reference substance of 5-HMF 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 5-HMF as an impurity in medicinal products.

^{−1}•L•cm^{−1}at 284 nm) may be used to quantify 5-HMF as an impurity in medicinal products containing glucose. For a specific medicinal product, a full validation of the analytical procedure of the 5-HMF determination is required taking into account the composition of this product.

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 5-HMF concentrations based on the colored product which is formed as a result of the reaction between 5-HMF,

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 5-HMF 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 5-HMF spectral characteristics, linearity, intermediate precision, range, detection limit, quantitation limit of the analytical procedure of the 5-HMF determination and estimate the possibility of using molar absorption coefficient ԑ for 5-HMF 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 5-HMF spectral characteristics and compare them with literature data as well.

5-HMF (analytical standard, batch number: BCBR3219V) was purchased from Sigma-Aldrich (USA). It was dissolved in purified water before analysis. As stock solutions were ones with 5-HMF 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 5-HMF determination was employed.

The five aqueous 5-HMF solutions with the concentrations in the range of 2–10 mg/L were prepared using purified water as a solvent. The 5-HMF concentrations were calculated using the known values of the molar absorption coefficient ԑ for 5-HMF at the wavelength of maximum absorption at 283-284 nm according to the following formula:

where C – 5-HMF concentration in mg/L, A – solutions absorbance at an absorption maximum of 283–284 nm, M.m. – molar mass of 5-HMF (126 g/mol), ԑ – molar absorption coefficient.

Spectrophotometer Hitachi U-2810 (Hitachi High-Technologies Corporation, Japan) was used. A 1-cm quartz cell was used over the range of 200 to 350 nm.

It was shown in a published paper (

Spectra of 5-HMF at the concentrations of 1.97 mg/L, 3.94 mg/L, 5.91 mg/L, 7.88 mg/L, and 9.85 mg/L (

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 _{0}) for the calibration line was 0.0037.

Correlation between absorbance at 284 nm and 5-HMF concentration in the first experiment.

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 5-HMF as a reference substance. Fig. _{0} was 0.00662. The second study confirmed the spectral characteristic of 5-HMF, namely the two absorption maxima at the wavelengths of 229–230 nm and 284 nm.

Correlation between absorbance at 284 nm and 5-HMF concentration in the second experiment.

The detailed spectral characteristic of 5-HMF in the first and second experiments are given in Tables

Detailed spectral characteristic of 5-HMF (01 August 2016).

5-HMF 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 5-HMF (10 December 2016).

5-HMF 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, one-tailed 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 y-intercepts did not exceed their confidence interval that confirms the correctness of using the molar absorption coefficient to determine 5-HMF content.

We also experimentally determined molar absorption coefficient for 5-HMF at 229–230 nm. To the best of our knowledge, only one available publication provides the value of the molar absorption coefficient for 5-HMF at 228 nm, which is equal to 3000 L•mol^{-1}•cm^{-1}(

where A – solutions absorbance at the first absorption maximum, M.m. – molar mass of 5-HMF (126 g/mol), C – 5-HMF concentration in mg/L.

The molar absorption coefficient of 5-HMF 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 5-HMF 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 5-HMF at an absorption maximum of 283–284 nm were used for the estimation of the analytical procedure recovery:

17000 mol −1•L•cm −1 (Kjellstrand et al. 2004);

22700 mol −1•L•cm −1 (Zhang et al. 2013);

16830 mol −1•L•cm −1 (Polydextrose).

The average recovery values for the molar absorption coefficients were calculated for each experiment (Tables ^{-1}•L•cm^{-1} from USP-NF (Polydextrose), the mean recovery is equal to 95.5%. Such a deviation from 100% is acceptable based on the criterion of the State Pharmacopeia of Ukraine (∆_{As} ≤ 5%). However, for a value of 17000 mol^{-1}•L•cm^{-1}(^{-1}•L•cm^{-1} (^{-1}•L•cm^{-1}) are unsuitable for the quantitative determination of 5-HMF as an impurity in medicinal products.

Limit of detection (LoD) and limit of quantification (LoQ) were calculated from the SD of y-intercept and the slope of the calibration lines (

Thus, the developed direct spectrophotometric method of the 5-HMF 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 5-HMF 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 5-HMF 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 5-HMF, provided that validation of the analytical procedure for each medicinal product is studied.

Co-author Natalia Hudz is grateful to the International Visegrad Fund for providing scholarship for studies related to solutions for dialysis therapy.

^{nd}edn). 416 pp.