INTRODUCTION:

Oxaliplatin, OXPt (trans-(R,R)-cyclohexane-1,2-diamine) oxalatoplatinum) belongs to platinum-containing anticancer drugs. OXPt has been approved for the treatment of colorectal cancer, showing efficacy in many cancer cell lines and tumors that are resistant to cisplatin and carboplatin^1-5. The therapeutic effect of OXPt in the treatment of pancreatic and stomach cancer, non-small cell lung cancer and breast cancer has been recorded^6-9.

OXPt is not only a standard option for the treatment of oncological diseases, but is used for adjuvant therapy in complete resection of stage III primary colon cancer. Pure racemic mixtures of OXPt are rather difficult to obtain. In the future, they are the basis for obtaining enantiomers. The nature of the substituents, the stereochemistry at the C(4) atom of the cyclohexane ring significantly contribute to the cytotoxicity of OXPt^10-14.

Therefore, two ways of obtaining selective racemates with four isomers are important: 1) the synthetic way of substances with equatorial substituents in the C(4) position of cyclohexane, 2) the construction of cyclohexane-1 - substituted derivatives of 2-diamine (substituents with a fixed configuration^15-18.

OXPt is more lipophilic than diaminoplatinum (II) complexes. The adducts it forms with the primary target DNA are differentially processed by cells. Due to the methylene links of the cyclohexane fragment, a non-polar region is formed on DNA, which contributes to different recognition and repair of OXPt and DNA adducts. The lipophilicity of OXPt is a result of the formation of the 1,2-diaminocyclohexane ligand compared to cis- and carboplatin. Lipophilic properties of OXPt contribute to its toxicity, changes in absorption by cells. The diamine ligand and the stereochemical properties of the ligand play an important role in the cytotoxic profile of OXPt^19-23. Platinum complexes with amino groups in the trans position show better cytotoxic and antitumor activity when compared with the activity of the isomer–cis-(R,S)-cyclohexane-1,2-diamine) oxalatoplatinum. The trans-(R,R)-isomer is a more potent antitumor agent compared to the trans-(S,S)-congener.

Due to the complexity of the synthesis of the OXPt substance, the separation of isomers and the purification process, it is possible to foresee the formation of intramolecular bonds between the molecules of the substance due to free functional groups and the formation of by-products during synthesis, the degradation of molecules. In addition, OXPt is a toxic substance.

Therefore, the quality control of the OXPt substance should be carried out by the high-tech HPLC method, since the State Pharmacopoeia of Ukraine does not regulate the analysis of this substance^24-29. Eur.Ph. regulates the analysis of specific impurities in the composition of the OXPt substance by the LC method³⁰. The high quality of the OXPt substance is important for protecting the health and life of cancer patients.

The actual task of the experimental research is the adaptation with possible modification of the chromatographic conditions when studying the OXPt substance by the HPLC method, the methods of sample preparation when performing studies by the HPLC method, which will allow implementing the HPLC method in the practice of pharmaceutical analysis of OXPt and its structural analogues and obtaining correct conclusions about the quality of the studied samples.

AIM OF THE RESEARCH:

Adaptation of chromatographic conditions with possible modification for HPLC analysis and research methods for the substance OXPt, which will allow correct conclusions to be drawn regarding the quality of its studied samples.

materials aNd methods:

Samples of the substance OXPt, pharmacopoeial standard samples of SPU OXPt and specified impurity C; HPLC, Agilent 1260 Infinity II chromatograph with UV detector, INERTSIL ODS-3V column, 250x4.6x5; purity reagents and solvents for HPLC; computer analysis using the OpenLab CDS program.

Results:

Eur.Ph. regulates the analysis of OXPt substance²⁰. Identification of accompanying impurities is carried out using the LC method (2.2.29). The recommended mobile phase is a mixture of solvents: ethanol R - methanol R (30:70, V/V). UV detection is performed by: imp. A at 205nm, imp. B at 215nm, imp. C at 210 nm, imp. D at 254nm. The temperature of the column is 40°C. The specified impurities include impurities A, B, C, D, impurity E – oxalic acid.

Impurity limits are set: imp. A - 0.15%, imp. B- 0.15%, imp. D - 0.15%. Total limit of impurities (except imp. D) – 0.30%. According to Eur.Ph. Rt: OXPt – 14min, imp. A - 4.7min, imp. B – 4.3min, imp. D – 16min, imp. E - 6.4min.

Imp. D (S,S-enantiomer of oxaliplatin) is detected by the LC method (2.2.29): 30mg of the substance is dissolved in methanol R, brought to a volume of 50 ml with methanol R.

As a mobile phase, a mixture is used: acetonitrile R – ammonia solution conc. R – methanol R – methylene chloride R (10:20:40:40, V/V/V/V).

In this work, the study of Oxaliplatin substance, based on the pharmacopoeial methods described in the literature, is performed and described for the first time.

Experimental research was performed on an Agilent 1260 Infinity II chromatograph with UV detection at 254nm, using a column - INERTSIL ODS-3V, 0.25x4.6x5 with a temperature of 40˚С. Chromatography time – 56min.

Chromatography conditions: flow - 0.3ml/min; injection volume – 20μl; mobile phase: ethanol R – methanol R (30:70, V/V); or in modified conditions: flow – 1.5 ml/min; mobile phase: acetonitrile - formic acid (0.01 M) (10:90; V/V); working solutions of OXPt (100.0μg/ml) were prepared by dissolving 10.0mg of the substance in 100ml of purified water. Standard solutions were used to prepare sample calibration curves.

To determine extraneous impurities by HPLC, the following reagents were used: acetonitrile (purity for HPLC), methanol (purity for HPLC), ethanol (purity for HPLC).

The method of preparing the test solution: 30mg of the substance is dissolved in methanol R, and the volume is brought to 50ml with the same solvent.

The method of preparation of the comparison solution (a): 5.0mg of substance OXPt with imp. D standard is dissolved in methanol R, and the volume is brought to 100.0ml with the same solvent.

The method of preparation of the comparison solution (b): 15ml of the comparison solution (a) is brought up to 50ml with methanol R.

The method of preparation of the comparison solution (c): dissolve 75.0mg of the substance in methanol R, make up to a volume of 100.0ml with the same solvent.

The method of preparation of the comparison solution (d): 5ml of the comparison solution (c) is brought to a volume of 100.0ml with with the same solvent.

Computer analysis using the OpenLab CDS program.

After the studying of Eur.Ph. standard samples, comparison solutions, solutions of test samples, the following results were obtained (tab. 1, 2; fig. 1,2).

Table 1. Solutions of standard substances.

	Standard (a)		Standard (b)		Imp.C specified
	RT	Area	RT	Area	RT
	7.443	59.837	7.544	62.047	3.620
	7.498	61.500	7.439	63.333	3.638
	7.401	59.467	7.421	62.690	3.629
Average	7.447	60.268	7.468	62.690	3.629

Standard solutions: OXPt, Standards (а, b):

- The value of Rt is in the interval 7.401-7.544 min;

- The average value of Rt standards is in the interval 7.447-7.468min;

- The plane of the peak on the chromatogram ranges from 59.467 to 63.333;

- The average value of the peak plane on the chromatogram of the standard sample is 60.268-62.690;

- RSD Rt (<2.0%) 0.30-0.38%;

- RSD Ar (<2.0%) 1.45-1.80%.

Test solutions: OXPt (1,2):

- The value of Rt is in the interval 7.422-7.540 min;

- The average value of Rt is in the interval 7.457-7.465 min;

- The plane of the peak on the chromatogram ranges from 58.467 to 62.432;

- The average value of the peak plane on the chromatogram of the sample is 59.289-62.102.

Figure 1. OXPt chromatogram of the standard sample (a) with specified impurity C.

Figure 2. OXPt chromatogram of the test sample 1 with specified impurity C and unspecified impurity 1, inadmissible unidentified impurities.

Specified impurity C was found in the tested samples 1, 2. Unspecified impurity 1

– trans-(1,2-cyclohexanediamine-N,N)dichloridoplatinum (II)) was found in the test sample 1. Inadmissible unidentified impurities were found: 1 – Rt 2.538min; 2 – Rt 4.345min, 3 – Rt 5.510 min.

DISCUSSIONS:

Thus, the test samples of the OXPt substance contain the specified impurity C (according to Eur.Ph.), unspecified impurity 1–trans-(1, 2-cyclohexanediamine-N,N) dichloridoplatinum (II)), inadmissible unidentified impurities 1, 2, 3 – impurities, the presence of which is not allowed in the substance.

In addition, the presence of non-specific and unacceptable impurities in the composition of the OXPt substance affects changes in the chromatographic pattern:

OXPt, standard (а, b):	OXPt, samples 1, 2:
The value of Rt is in the interval 7.401-7.544 min	The value of Rt is in the interval 7.422-7.540 min
Offset values by 0.053-0.28%
The average value of Rt standards is in the interval 7.447-7.468 min	The average value of Rt is in the interval 7.457-7.465 min
Offset values by 0.040-0.134%
The plane of the peak on the chromatogram ranges from 59.467 to 63.333	The plane of the peak on the chromatogram ranges from 58.467 to 62.432
Offset values by 1.422-1.681%
The average value of the peak plane on the chromatogram of the standard sample is 60.268-62.690	The average value of the peak plane on the chromatogram of the standard sample is 59.289-62.102
Offset values by 0.937-1.624%

The obtained results allow us to conclude that the studied OXPt substances do not meet the requirements of pharmacopoeial analysis (according to Eur.P.), as they contain non-identified impurities, and are subject to thorough purification and further investigation of the structure of unidentified impurities by mass spectrometry.

findings:

1. The conditions of chromatographic research and the methods of preparing samples for analysis by HPLC of OXPt substance were modified, namely, modifications were proposed: working solutions of OXPt (100.0 μg/ml) were prepared by dissolving 10.0 mg of the substance in 100 ml of purified water; mobile phase: acetonitrile - formic acid (0.01 M) (10:90; V/V); the flow rate is 1.5 ml/min, as a result of which the chromatograms of the standard and test samples were compared.

2. Using the HPLC method, it was found that in the composition of the studied samples, in addition to the specified impurity C, there are an accompanying impurities with an unidentified structure, which must be investigated by mass spectrometry.

3. Chromatography under the proposed conditions using the HPLC method (modified working solutions, mobile phase, flow rate) revealed an unidentified accompanying impurity in the substance OXPt, which confirms the need for additional purification of the substances under study.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

THANKS TO:

The authors thank Zaporizhzhia State Medical and Pharmaceutical University, Bogomolets National Medical University, State Agency «O.M. Marzeev Institute of Public Health of the National Academy of Medical Sciences of Ukraine» for its kind support during research.

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Received on 13.05.2025 Revised on 16.09.2025

Accepted on 29.11.2025 Published on 05.06.2026

Available online from June 06, 2026

Research J. Pharmacy and Technology. 2026;19(6):2504-2508.

DOI: 10.52711/0974-360X.2026.00358

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

	Test substance 1		Test substance 2		Imp. C, specified	Imp. 1 – trans-(1,2-cyclohexanediamine-N,N) dichloridoplatinum (II)), unspecified
	RT	Area	RT	Area	RT	RT
	7.454	58.836	7.540	61.999	3.621	6.768
	7.485	60.566	7.422	62.432	3.625	6.822
	7.433	58.467	7.434	61.876	3.627	6.898
Average	7.457	59.289	7.465	62.102	3.624	6.829