Biomedical Research

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Research Article - Biomedical Research (2019) Volume 30, Issue 4

Spectrophotometric determination of cisplatin, carboplatin and oxaliplatin in pure and injectable dosage forms

Muhammad Munawar Hayat1*, Muhammad Sohail1, Muhammad Ashraf2

1Primary and Secondary Healthcare Department, Government of the Punjab, Lahore, Pakistan

2Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur, Paki stan

*Corresponding Author:
Muhammad Munawar Hayat
Primary and Secondary Healthcare Department
Lahore
Pakistan

Accepted Date: May 23, 2019

DOI: 10.35841/biomedicalresearch.30-19-244

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Abstract

Objective: A simple and reproducible spectrophotometric method was developed for the estimation of cisplatin, carboplatin and oxaliplatin in pure and injectable dosage forms. Method: The method is based on the reaction of platinum drug with 1,10-phenanthrolene in the presence of ferric chloride, which formed a red coloured complex that exhibited maximum absorption at 510 nm. The reaction is selective for platinum based drugs and obeys the Beer’s law for cisplatin from 0.25-6.0 μg, carboplatin 7.5-180 μg and oxaliplatin 0.5-12.0 μg. The accuracy of the developed method for cisplatin, carboplatin and oxaliplatin was 98.6%, 100.3% and 101.1%, whereas RSD (%) is of 1.1, 1.3 and 1.8, respectively. Result: The standardization and validation of analytic method was evaluated by the parameters like linearity, accuracy, precision and sensitivity. Conclusion: The method is useful for the determination of these drugs in pure and dosage forms.

Keywords

Cisplatin, Carboplatin, Oxaliplatin, 1-10, Phenanthrolene, Spectrophotometry

Introduction

The platinum based drugs are famous family of antiproliferative agents and were identified by Rosenberg and co-workers in 1965. They act like alkylating agents by cross linking DNA. The parent drug cisplatin is widely used platinum based chemotherapeutic agent, whereas the carboplatin and oxaliplatin have improved toxicity profile and provide better results in germ cell, ovarian and colorectal cancers [1].

Cisplatin (Figure 1) belongs to an alkylating class of anticancer drugs and is the first member of platinum containing drugs used as antineoplastic in early 1970’s. It is a divalent inorganic, water soluble platinum containing complex and is widely used due to its potent effects in the treatment of ovarian, testicular, head and neck tumor, cervical cancer and small cell lung carcinoma [2,3]. Carboplatin (Figure 1) was approved for clinical use in 1989. The mechanism of action and resistance of carboplatin are similar to cisplatin, but differs significantly in chemical, pharmacokinetic and toxicological properties [4]. Carboplatin is less reactive and well tolerated and is used as intravenous infusion to treat patients who are unable to tolerate cisplatin as front-line treatment for ovarian cancer. It is currently approved by FDA for use in the treatment of advanced ovarian and lung cancer in combination with paclitaxel or cyclophosphamide [5]. Oxaliplatin (Figure 1) is the most recent drug approved by FDA in 2002. It exhibits a wide range of antitumor activities that differ from other platinum drugs. It also suppresses the enzyme thymidylate synthetase which is also target enzyme of 5- fluorouracil, which is the reason of synergy of two drugs. It is used to treat colorectal cancers in combination with 5-fluorouracil and leucovorin [6].

biomedical-research-platinum

Figure 1: Structures of platinum based drugs.

Several methods for the determination of platinum based drugs include HPLC with UV detection [7,8], LC-MS [9] and ICPMS [10,11]. These platinum based drugs have also been determined by spectrophotometric method [12-14]. The previously published methods included derivitization of cisplatin or demanded costly equipment [9-11]. This work demonstrates direct and simultaneous spectroscopic method for the determination of platinum based drugs by spectrophotometric method.

Materials and Methods

Apparatus

A UV-visible double beam spectrophotometer (Shimadzu 1601, Japan) and 96-well plate reader, Synergy HT, USA were used to measure the absorbance.

Chemicals and drugs

Cisplatin, carboplatin and oxaliplatin were kindly gifted by Pharmedic Laboratories Pvt. Ltd. Lahore, (Pakistan). Ferric chloride, 1,10-phenanthrolene, phosphoric acid and de-ionized double distilled water were of analytic grade. Commercial injectable dosage forms of cisplatin, carboplatin and oxaliplatin were purchased from local market.

Preparation of 1,10-phenanthroline solution: 1,10- Phenanthroline monohydrate (0.4 gram) was dissolved in 100 ml double distilled water to form 0.4% solution.

Preparation and standardization of phosphoric acid solution: A fixed volume (25 μl) of phosphoric acid of different molarity was mixed with cisplatin (150 μg/ml), carboplatin (450 μg/ml) and oxaliplatin (150 μg/ml) followed by the addition of 40 μl of ferric chloride (0.5%) and 100 μl of 0.2% or saturated solution (0.4%) of 1, 10-phenanthrolene solution and absorbance was measured at 510 nm.

Ferric chloride solution: Ferric chloride (0.5 gram) was dissolved in 100 ml double distilled water to constitute a 0.5% solution.

Standard stock solution preparation: Stock solutions of cisplatin and oxaliplatin (1000 μg/ml) were prepared by dissolving accurately weighed 10 mg in double distilled water and making the volume to 10 ml in standard volumetric flask. Carboplatin was prepared by dissolving accurately weighed 30 mg in 10 ml of double distilled water to get a concentration of 3.0 mg/ml. Working solutions of lower concentration was prepared by further dilution of standard stock solutions with de-ionized double distilled water.

Sample preparation: A commercial brand of cisplatin injection (Unistin-Korea), carboplatin injection (Cytocarb- Cipla) and oxaliplatin injection (Oxilitin- Pharm Evo) were chosen.

Results

Absorption spectrum of coloured complex

The absorption spectra of cisplatin, carboplatin and oxaliplatin are shown in Figures 2-5.

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Figure 2: Calibration curve of cisplatin, carboplatin and oxaliplatin.

biomedical-research-spectrum

Figure 3: Spectrum of cisplatin (λmax 510 nm)

biomedical-research-carboplatin

Figure 4: Spectrum of carboplatin (λmax 510 nm).

biomedical-research-oxaliplatin

Figure 5: Spectrum of oxaliplatin (λmax 510 nm).

Chemistry of coloured complex

The present method was based on the formation of red coloured complex with ferric chloride after its reduction to ferrous form. This complex resulting from platinum based drugs may be due to the fact that each of the two nitrogen atoms has an unshared pair of electrons that can be shared with Fe (II) ion formed by the reaction of platinum with Fe (III). Three molecules of 1,10-phenanthroline attach themselves by means of dative bonds to form ferroin complex.

Method Development

A spectroscopic method was developed in pure and applied on injectable brands of platinum based drugs as follows.

Procedure

Aliquots of working standard solutions (cisplatin, carboplatin and oxaliplatin) of 5, 10, 20, 40 and 60 μl were added in triplicate in 96 well-plates with 40 μl of 0.5% ferric chloride and 100 μl of 0.2% or saturated solution (0.4%) of 1,10- phenanthrolene. Contents were incubated at 60ºC for 45 minutes, cooled at room temperature followed by addition of 25 μl of 15 mM phosphoric acid. Finally, the solution was mixed thoroughly and absorbance was measured at 510 nm against the blank prepared under same identical conditions and calibration curves were constructed (Figure 2). The system was attached with computer software (Gen 5) to analyze and calculate data (Tables 1 and 2).

Cisplatin
(µg)
Abs. of cisplatin at
510 nm
Carboplatin
(µg)
Abs. of carboplatin at 510 nm Oxaliplatin
(µg)
Abs. of oxaliplatin at 510 nm
0 0.543 0 0.543 0 0.543
0.25 0.563 7.5 0.591 0.5 0.564
0.5 0.589 15 0.638 1 0.583
1 0.609 30 0.703 2 0.599
2 0.701 60 0.898 4 0.679
3 0.749 90 1.091 6 0.751
4 0.828 120 1.276 8 0.808
5 0.905 150 1.493 10 0.878
6 0.987 180 1.717 12 0.952

Table 1: Spectrophotometric determination of platinum based drugs.

Sample (μl) Water
(μl)
0.5 % FeCl3
 (μl)
0.4 % 1,10-Phenanthrolene (μl)   15 mM
H3PO4
(μl)
0 60 40 100   25
2.5 57.2 40 100   25
5 55 40 100   25
10 50 40 100   25
20 40 40 100   25
30 30 40 100   25
40 20 40 100   25
50 10 40 100   25
60 0 40 100 Heated at 60ºC for 45 minutes 25

Table 2: Absorbance (510 nm) of cisplatin, carboplatin and oxaliplatin vs concentration (μg /ml).

Method validation

The developed spectrophotometric method was standardized validated with following parameters.

Linearity and range: Linearity of the method was determined by constructing calibration curves for cisplatin, carboplatin and oxaliplatin (Figure 2). The standard stock solution of cisplatin, carboplatin and oxaliplatin (1.0 μg/ml) were prepared in distilled water (Table 3).

Drug Slope Intercept r2
Cisplatin 0.072 0.544 0.997
Carboplatin 0.006 0.52 0.996
Oxaliplatin 0.33 0.543 0.998
Table 3: Standard curve parameters for platinum based drugs (mean values, n=3).

Sensitivity: The LOQ values of cisplatin, carboplatin and oxaliplatin were 0.25, 7.5 and 0.5 μg/ml and LOD values of cisplatin, carboplatin and oxaliplatin were 0.12, 3.75 and 0.25 μg/ml, respectively (Table 4).

Parameters Cisplatin Carboplatin Oxaliplatin
Colour Red complex Red complex Red complex
λmax 510 nm 510 nm 510 nm
Range 0.25-6.0 µg 7.5-180 µg 0.5-12 µg
LOD 0.12 µg/ml 3.75 µg/ml 0.25 µg/ml
LOQ 0.25 µg/ml 7.5 µg/ml 0.5 µg/ml
r-square 0.9974 0.9979 0.9985
slope 0.0724 0.0065 0.0337
intercept 0.5443 0.5209 0.5433
Accuracy 98.6 100.3 101.1
RSD (%) 1.1 1.3 1.8

Table 4: Parameters and sensitivity (mean values, n=3).

Accuracy: Accuracy was determined by selecting low, medium and high concentrations of cisplatin, carboplatin and oxaliplatin in triplicate. The mean values (low, medium and high) of accuracy for pure drugs were given in Tables 5 and 6 for injectable dosage forms of cisplatin, carboplatin & oxaliplatin were given in Table 7.

Drug Parameters LC MC HC
Cisplatin Mean 0.2427 2.925 5.84
S.D. 0.007 0.079 0.121
N 9 9 9
Nominal 0.25 3 6
% CV 2.986 2.69 2.069
% Accuracy 97.07 97.51 97.33
Carboplatin Mean 7.2812 97.267 176.061
S.D. 0.1413 0.7886 1.085
N 9 9 9
Nominal 7.5 100 180
% CV 1.9407 0.8108 0.6163
% Accuracy 97.08 97.27 97.81
Oxaliplatin Mean 0.4892 5.891 11.809
S.D. 0.0125 0.1331 0.322
N 9 9 9
Nominal 0.5 6 12
% CV 2.5546 2.2598 2.7264
% Accuracy 97.84 98.18 98.41

Table 5: Determination of between-batches accuracy.

Drug Parameters LC MC HC
Cisplatin Mean 0.2427 2.915 5.861
S.D. 0.004 0.085 0.166
N 6 6 6
Nominal 0.25 3 6
% CV 1.535 2.922 2.831
% Accuracy 97.07 97.17 97.68
Carboplatin Mean 7.2788 97.093 175.336
S.D. 0.148 0.553 0.396
N 6 6 6
Nominal 7.5 100 180
% CV 2.027 0.569 0.226
% Accuracy 97.05 97.09 97.41
Oxaliplatin Mean 0.497 5.8512 11.823
S.D. 0.01 0.129 0.159
N 6 6 6
Nominal 0.5 6 12
% CV 2.101 2.211 1.342
% Accuracy 99.37 97.52 98.52

Table 6: Determination of with-in batch precision.

Serial No. Sample File name Absorbance
at 510 nm
 Quantity quoted in dosage forms
(µg/ml)
Quantity determined
 (µg/ ml)
Accuracy
(%)
1 Cisplatin
Brand (Unistin)
Unistin 1 0.901 5 4.94 98.8
2 Unistin 2 0.898 5 4.9 98
3 Unistin 3 0.904 5 4.99 99.8
4 Unistin 4 0.894 5 4.85 97
5 Unistin 5 0.902 5 4.96 99.2
Mean       4.928 98.6
SD       0.054 -
RSD       1.1 -
N       5 5
1 Carboplatin
brand (Cytocarb)
Cytocarb 1 1.187 100 101.02 101
2 Cytocarb 2 1.171 100 98.66 98.6
3 Cytocarb 3 1.194 100 102.05 102
4 Cytocarb 4 1.183 100 100.43 100.4
5 Cytocarb 5 1.176 100 99.39 99.4
Mean       100.31 100.3
SD       1.334 -
RSD       1.3 -
N       5 5
1 Oxaliplatin brand (Oxalitin) Oxalitin 1 0.881 10 9.94 99.4
2 Oxalitin 2 0.879 10 9.89 98.9
3 Oxalitin 3 0.889 10 10.17 101.7
4 Oxalitin 4 0.894 10 10.32 103.2
5 Oxalitin 5 0.891 10 10.23 102.3
Mean       10.11 101.1
SD       0.187 -
RSD       1.8 -
N       5 5

Table 7: Determination of concentration of the injectable brands of cisplatin, carboplatin and oxaliplatin by forecast formula (y = a + b x).

Precision: Precision of the method was performed at three different concentrations (low, medium and high) in the range in triplicates. The CV with-in-batch and between batches for low, medium and high concentrations of cisplatin, carboplatin and oxaliplatin were given in Tables 5 and 6. The values were less than 3% and within the range of FDA guidelines.

Discussion

The present study was designed to develop a spectrophotometric method for determination of platinum based drugs. Already there was a single method only for determination of oxaliplatin 14 but no single method for simultaneous determination of cisplatin, carboplatin and oxaliplatin. Hence, a direct, simple and reproducible spectrophotometric method was developed for the estimation

The method is based on the reaction of platinum with saturated solution (0.4%) of 1,10-phenanthrolene in the presence of ferric chloride, which forms a red coloured complex on heating at 60ºC for 45 minutes and added 15 mM phosphoric acid.

The formed complex exhibited maximum absorption at 510 nm. This method was successfully applied on commercial brand of cisplatin injection (Unistin-Korea), carboplatin injection (Cytocarb-Cipla) and oxaliplatin injection (Oxilitin- Pharm Evo). The method had wide linear range with good accuracy and precision (Tables 2-4).

The applicability of the developed method for assay of injectable pharmaceutical dosage form was examined and results were summarized in Table 7. The results are highly reproducible.

Conclusion

The present spectrophotometric method is found to be economical, direct, more sensitive and unique method for the simultaneous analysis of platinum based drugs (cisplatin, carboplatin, oxaliplatin) in pure and injectable dosage forms. The statistical parameters show that method has importance in quality analysis of injectable dosage forms of platinum based drugs.

References