Analytical Method Development and Validation for the Estimation of Anticancer Drugs: A Comprehensive Review

CANCER

Cancer can be defined as a group of abnormal cells grow uncontrollably by disregarding the normal rules of cell division. Normal cells are constantly subject to signals that dictate whether the cell should divide, differentiate into another cell or die. Cancer cells build a degree of autonomy from these signals, resulting in uncontrolled growth and proliferation. If this proliferation is allowed to continue and spread, it can become fatal. In fact, almost 90% of cancer-related deaths are because of tumor spreading which is called metastasis.

The prevailing theory, which underpins research into the genesis and treatment of cancer, is that normal cells are transformed into cancers as a result of changes in these networks at the molecular, biochemical and cellular level, and for each cell there is a finite number of ways this disruption can occur.

Figure 1.1 Clonal expansion

Cancer is a multi-gene, multi-step disease originating from single abnormal cell (clonal origin). Some cells in the tumor  undergo further  rounds  of mutations causing to the formation of malignant cells which lead to metastasis.

Figure 1.2 Clonal Expansion

Multiple           mutations         cause   a tumor mass subsequent mutations malignant tumor, which is broken by the basal membrane and spreads to distant locations.

Causes of cancer:

• Environment and influence of lifestyle
• infectious agents [viruses -hepatitis B virus (HBV), human papilloma virus (HPV), human immunodeficiency virus (HIV)]
• cancer-leading agents (carcinogens, mutagens)
• Age

Treatment:

There are a number of options available for treatment of cancer with the preferred choice of therapy dependent upon location and type of tumor and the disease stage to which the cancer has progressed. Preferred treatment options include surgery to remove discrete, localized tumors such as those found in prostate and breast cancers; radiation therapy using ionizing radiation to kill cancer cells and shrink tumors (useful in the treatment of most solid tumors as well as leukemia and lymphoma); and chemotherapy, the treatment of cancer cells with drugs to destroy them. Anticancer drugs are targeted towards cancerous cells in the human body. Since, cancerous cells are similar in nature and function to the other human cells, anti cancer drugs find it difficult to differentiate between normal cells and tumourous cells. Anti cancer activity is achieved by targeting at various stages of cell – division.

1.2       CLASSIFICATION OF ANTICANCER DRUGS

1.3       DRUG PROFILE2-8

Generic Name	Temozolomide
Description	White to light brown
Chemical Structure
IUPAC Name	3-methyl-4-oxoimidazol[5,1-d][1,2,3,5]tetrazine-8- carboxamide
Molecular Formula	C6H6N6O2
Molecular Mass	194.15 g/mol
Category	Anticancer (Antineoplastic agent, Alkylating)
Solubility	Soluble in Water, DMSO, Ethanol
pKa	Strongest acidic: 10.51 Strongest basic: -3.6
Storage	Store protected from light and moisture
Mechanism of Action	Temozolomide is an imidazotetrazine alkylating agent with antitumor activity that can be used orally. It undergoes rapid chemical conversion in the systemic circulation at physiologic pH to the active compound, MTIC. The cytotoxicity of MTIC is thought to be due primarily to alkylation at the O6 position of guanine with additional alkylation also occurring at the N7 position. Cytotoxic lesions that develop subsequently are thought to involve aberrant repair of the methyl adduct.

Generic Name	Capecitabine
Description	White to off-white crystalline powder
Chemical Structure
IUPAC Name	(1-(5-Deoxy-beta-D-ribofuranosyl)-5-fluoro-1,2-dihydro-2- oxo-4-pyrimidinyl)-carbamicacid pentyl ester
Molecular Formula	C15H22FN3O6
Molecular Mass	359.35 g/mol
Category	Anticancer
Solubility	Soluble in Water, DMSO, Methanol
pKa	Strongest acidic: 8.33 Strongest Basic: -3.6
Storage	Store  at  controlled  room  temperature,  protected  from moisture. Preserve in tight containers.
Mechanism                                of Action	Capecitabine is a prodrug that is selectively tumor-activated to its Cytotoxic moiety, fluorouracil, by thymidine phosphorylase, an enzyme found in higher concentrations in tumors compared to normal tissues or plasma. Fluorouracil is further metabolized to two active metabolites, 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and 5-fluorouridine triphosphate (FUTP), within normal and tumor cells. These metabolites cause cell injury by two different mechanisms. First, FdUMP and the folate cofactor, N5-10-methylenetetrahydrofolate, bind to thymidylate synthase (TS) to form a covalently bound ternary complex. This binding inhibits the formation of thymidylate from 2'-deoxyuridylate. Thymidylate is the necessary precursor of thymidine triphosphate, which is essential for the synthesis of DNA, therefore a deficiency of this compound can inhibit cell division. Secondly, nuclear transcriptional enzymes can mistakenly incorporate FUTP in place of uridine triphosphate (UTP) during the synthesis of RNA. This metabolic error can interfere with RNA processing and protein synthesis through the production of fraudulent RNA.

1.3.3   Drug profile of gemcitabine hydrochloride

Generic name	Gemcitabine HCl
Description	White or almost white powder
Chemical structure
IUPAC name	4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5- (hydroxymethyl)oxolan-2-yl]-1,2-dihydropyrimidin-2-one
Formula	C9H11F2N3O4
Molecular mass	299.69 g/mole
Category	Anticancer
Solubility	Soluble in water, slightly soluble in methanol, practically in soluble in acetone
PKa	3.6
Storage condition	If the substance is sterile, store in a sterile, airtight, tamperproof container.
CAS Number	122111-03-9
Mechanism of action	Gemcitabine inhibits thymidylatesynthease, leading to inhibition of DNA synthesis and cell death. Gemcitabine is a prodrug so activity occurs as a result of intracellular conversion to two active metabolites, gemcitabine diphosphate and gemcitabine triphosphate by deoxycitidine kinase. Gemcitabine diphosphate also inhibits ribonucleotidereductase, the enzyme responsible for catalyzing synthesis of deoxynucleoside triphosphates required for DNA synthesis. Finally, Gemcitabine triphosphate (diflurorodeoxycytidine triphosphate) competes with endogenous deoxynucleoside triphosphates for incorporation into DNA.

1.3.4    Drug profile of oxaliplatin 12-15

Generic name	Oxaliplatine
Description	White/almost white, crystalline powder
Chemical structure
IUPAC name	[SP-4-2-(1R-trans)]-(1,2-cyclohexanediamine- N,N‘)[ethanedioato(2-)-O,O‘]platinum.
Formula	C8H14N2O4 Pt
Molecular mass	397.29g/mol
Category	Anticancer
Solubility	Slightly soluble in water, Very slightly in methanol, Practically insoluble in anhydrous ethanol
PKa	5.88
Storage condition	Store at room temperature.
CAS Number	61825-94-3
Mechanism of action	Oxaliplatin undergoes nonenzymatic conversion to active derivatives via displacement of the labile oxalate ligand. After activation, oxaliplatin binds preferentially to the guanine and cytosine moieties of DNA, leading to cross- linking of DNA, thus inhibiting DNA synthesis and transcription.

1.4       ADVANTAGE OF ANTICANCER COMBINATION16

• Temozolomide and oxaliplatin is an active agent in metastatic pancreatic endocrine carcinomas. In vitro data indicate that the combination of Capecitabine and Temozolomide and gemcitabin HCL and Oxaliplatin is synergistic for induction of apoptosis in neuroendocrine tumor cell lines.
• Early results from an ongoing phase II clinical trial have shown that a new chemotherapy combination, may be an effective second-line treatment option for patients with a neuroendocrine tumor that has spread to other parts of the body, even for tumors that haven‘t responded to other standard (commonly used) treatments.
• A second-line treatment is given if the first treatment does not work, starts but then stops working, or causes serious side effects. These combination, which are given in a specific order—first and second—based on research that showed this might be more effective than giving both drugs at the same time.
• The benefits of this treatment appear to be long-lasting.
• Researchers believe that this combination may eventually replace all other second-line therapies for metastatic neuroendocrine tumors.

1.5       DEVELOPMENT OF ANALYTICAL METHOD

A ‗regulatory analytical procedure‘ is used to evaluate a defined characteristic of the drug substance or drug product. An ‗alternative analytical procedure‘ is proposed by the applicant for use other than regulatory analytical procedure. A ‗stability-indicating assay‘ is a validated quantitative analytical procedure that can detect the changes with time in the pertinent properties of the drug substance and drug product. A stability-indicating assay accurately measures the active ingredients, without interference from degradation products, process impurities, excipients, or other potential impurities31. Stability testing forms an important part of the process of drug product development. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and enables recommendation of storage conditions, retest periods, and shelf lives to be established. The two main aspects of drug product that play an important role in shelf life determination are assay of active drug, and degradants generated, during the stability study. The assay of drug product in stability test sample needs to be determined using stability indicating method, as recommended by the International Conference on Harmonization (ICH) guidelines16 and USP 2617.

The modern methods of choice for quantitative analysis are HPLC, GC, and HPTLC, which are highly sophisticated. Chromatographic methods are commonly used in regulatory laboratories for the qualitative and quantitative analysis of drug substances, drug products, raw materials and biological samples throughout all phases of drug development, from research to quality control. High performance liquid chromatography18 (HPLC) is the fastest growing analytical technique for the analysis of drugs. Its simplicity, high specificity, and wide range of sensitivity make it ideal for the analysis of many drugs in both dosage forms and biological fluids. The rapid growth of HPLC has been facilitated by the development of reliable, moderately priced instrumentation and efficient columns. High performance thin-layer chromatography19 (HPTLC) is a classical separative technique that has employed wide spread popularity particularly in the analysis of complex mixtures of natural origin. Now-a-days HPTLC is becoming a routine analytical technique due to its advantages of low operating cost, high sample throughput, and need for minimum sample clean-up. The major advantage of HPTLC is that several samples can be run simultaneousdy using a small quantity of mobile phase unlike HPLC, thus lowering analysis time and cost per analysis.

1.5.1    Validation of analytical method 20-23

As defined by the USP, method validation provides an assurance of reliability during normal use, and is sometime referred to as —the process of providing documented evidence that the method does what it is intended to do.‖ The objective of validation of an analytical method is to demonstrate that the procedure, when correctly applied, produces results that are fit for purpose. To be fit for the intended purpose, the method must meet certain validation characteristics. Typical validation characteristics, which should be considered, are: selectivity (specificity), linearity, range, accuracy, precision, limit of detection, limit of quantitation, ruggedness, robustness and system suitability testing.

1.5.1.1 Selectivity (Specificity)

Selectivity of a method refers to the extent to which it can determine particular analyte(s) in a complex mixture without interference from other components in the mixture. The terms selectivity and specificity have often been used interchangeably. The term specific generally refers to a method that produces a response for a single analyte only, while the term selective refers to a method that provides responses for a number of chemical entities that may or may not be distinguished from each other. If the response is distinguished from all other responses, the method is said to be selective. Since very few analytical methods respond to only one analyte, the use of the term selectivity is more appropriate than specificity. The selectivity of the analytical method must be demonstrated by providing data to show the absence of interference peaks with regard to degradation products, synthetic impurities and the matrix (excipients present in the formulated product at their expected levels). The selectivity of chromatographic methods may be assessed by examination of peak homogeneity or peak purity test to show that the analyte chromatographic peak is not attributable to more than one component. Degradation information obtained from stress studies (e.g., products of acid and base hydrolysis, thermal degradation, photolysis and oxidation) for the drug substance and for the active ingredient in the drug product should be provided to demonstrate the specificity of the assay and analytical procedures for impurities. The stress studies should demonstrate that impurities and degradants from the active ingredient and drug product excipients do not interfere with the quantitation of the active ingredient. Stress studies are described in various FDA guidances relating to the stability of drug products.

1.5.1.2 Linearity

The linearity is the ability of analytical procedure to produce test results which are proportional to the concentration (amount) of analyte in samples within a given concentration range, either directly or by means of a well-defined mathematical transformation. Linearity should be determined by using a minimum of six standards whose concentration span 80 –120% of the expected concentration range. The linearity of a method should be established by visual inspection of a plot of analytical response as a function of analyte concentration. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of the regression line by the method of least squares. In some cases, the test data may need to be subjected to a mathematical transformation prior to regression analysis. Reports submitted must include the slope of the line, intercept and correlation coefficient data. The measured slope should demonstrate a clear correlation between response and analyte concentrations. The results should not show a significant deviation from linearity, which is taken to mean that the correlation coefficient, r > 0.99, over the working range (80-120 %).

1.5.1.3 Range

The specified range is normally derived from the linearity studies. The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical method has suitable levels of precision, accuracy and linearity.

1.5.1.5 Accuracy

The accuracy of an analytical method is defined as the degree to which the determined value of analyte in a sample corresponds to the true value. Accuracy may be measured in different ways and the method should be appropriate to the matrix. The accuracy of an analytical method may be determined by:

(A) Analyzing a sample of known concentration and comparing the measured value to the ‗true‘ value. However, a well characterized sample (e.g., reference standard) must be used.

(Β) Spiked-placebo (product matrix) recovery method:

In the spiked-placebo recovery method, a known amount of pure active constituent is added to formulation blank [sample that contains all other ingredients except the active (s)], the resulting mixture is assayed, and the results obtained are compared with the expected result.

(C) Standard addition method:

In the standard addition method, a sample is assayed, a known amount of pure active constituent is added, and the sample is again assayed. The difference between the results of the two assays is compared with the expected answer.

 In both methods (spiked-placebo recovery and standard addition method), recovery is defined as the ratio of the observed result to the expected result expressed as a percentage.

% Recovery calculated by formula;      

                             N (∑xy) - (∑x) (∑y)

% Recovery =                                    × 100

                             N (∑x²) - (∑x) ²

N = Number of observations

Y = Amount of drug found

X = Amount of standard drug added

1.5.1.5 Precision

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility.

(a)        Repeatability:

Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision.

(b)        Intermediate Precision:

Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc.

(c)        Reproducibility:

Reproducibility expresses the precision between laboratories. For these guidelines, a simple assessment of repeatability will be acceptable. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. A minimum of 5 replicate sample determinations should be made together with a simple statistical assessment of the results, including the percent relative standard deviation. The standard deviation (SD) is calculated from the following formula;

SD = ∑ (Xi - X) 2 /N-1

Xi = individual measurement in a set X = arithmetic mean of the set and

N = total number of replicated measurements taken in the set

Precision between different samples can be compared with relative standard deviation (RSD) as follows. The value of % RSD should be less than 2 %.

RSD = S/X

% RSD or coefficient of variance (CV) = (S/X) × 100

1.5.1.6 Limit of detection (LOD)

The lowest amount of an analyte in a sample that can be detected, but not necessarily quantitated as an exact value is termed as limit of detection. The LOD may be determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level (lowest calibration standard) at which the analyte can be reliably detected. The lowest calibration standard which produces a peak response corresponding to the analyte should be measured n times (normally 6-10). The average response (X) and the standard deviation (SD) calculated by formula;

LOD = X + (3 × SD).

1.5.1.7 Limit of quantitation (LOQ)

The lowest amount of the analyte in the sample that can be quantitatively determined with defined precision under the stated experimental conditions is termed as limit of quantitation. LOQ is a parameter of quantitative assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products or low levels of active constituent in a product. The LOQ may be determined by preparing standard solutions at estimated LOQ concentration. The solution should be injected and analyzed ‗n‘ times. The average response and the standard deviation should be calculated and the SD should be less than 2.0 %. If the SD exceeds beyond the critical value, a new standard solution of higher concentration should be prepared and the above procedure repeated. It can be calculated by formula;

 LOQ = X + (10 ×SD).

1.5.1.8 Ruggedness

Ruggedness is the degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions such as different laboratories, different analysts, different instruments, different lots of reagents, different elapsed assay times, different assay temperatures, different days etc. It is a measure of reproducibility of test results under nnrman `xpest d operational conditions from laboratory to laboratory and analyst to analyst. For methods developed for use on a wider scale, intensive testing for ruggedness is done through collaborative studies involving a number of laboratories.

1.5.1.9 Robustness

It is the measure of capability of analytical method to remain unaffected by small but deliberate variation in the method parameters and provides an indication of its reliability during normal range. Robustness testing is normally restricted to repetitively methods in the same laboratory. It means the method is repeatable when intentional variations such as change in concentration, use of different analyte, wavelength, use of different dilutions, change in column of the same type, small changes in mobile phase etc are introduced in the parameters internal to the method.

1.5.1.10 System suitability testing

System suitability testing is an integral part of many analytical procedures. They are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established for a particular procedure depend on the type of procedure being validated. For HPLC analysis, system suitability tests are

Number of theoretical plates (N) is a measure of column efficiency. It is calculated by formula

N = 16 (TR/W) 2          or   N = 5.54 (TR/W1/2)

TR = Retention time of the substance.

W = Width of peak measured by extrapolating the relatively straight sides to the base line for respective substances

W1/2 = Peak width at half height.

Resolution (R) is a function of column efficiency and is specified to ensure that closely eluting compounds are resolved from each other, to establish the general resolving power of the system, and to ensure that internal standards are resolved from the drug. Resolution can be calculated by formula;

R= 2 (TR2-TR1) / W1+W2

TR1 and TR2 = Retention of respective substances

W1 and W2 = Width of peak measured by extrapolating the relatively straight sides to the base line for respective substances

Relative standard deviation (RSD) is calculated from data of five replicate injections of analyte, if the requirement is less than 2.0 % or less; data from six replicate injections are used if RSD requirement is more than 2.0 %. The tailing factor (T), measure of peak symmetry, is unity for perfectly symmetrical peaks and its value increase as tailing becomes more pronounced. In some cases, value less than unity may be observed. As peak asymmetry increases, integration, and hence precision, becomes less reliable. Tailing factor calculated by formula;

T = W0.05 / 2F

W0.05 = Width of peak at 5 % height.

F = distance between the perpendicular dropped from the peak maximum and the leading edge of the peak at 5 % height

Asymmetry factor (AS) is defined as b/a, where ‗a‘ and ‗b‘ are the width at 10 % height for the front and back half of the peak, respectively.

CONCLUSION

The development and validation of analytical methods for anticancer drugs are fundamental processes that ensure the delivery of high-quality chemotherapy. This review confirms that RP-HPLC remains the most reliable and versatile technique for the estimation of antineoplastic agents due to its superior sensitivity and specificity. By strictly adhering to ICH Q2(R1) guidelines, researchers can establish methods that are not only accurate and precise but also robust enough for routine quality control in pharmaceutical industries. Furthermore, the integration of Stability-Indicating Assay Methods (SIAMs) provides a deeper understanding of drug degradation pathways, which is critical for determining shelf-life and storage conditions. As oncology moves toward personalized medicine, the evolution of these analytical techniques—transitioning from traditional UV methods to advanced LC-MS/MS and UPLC—will play a vital role in the future development of potent and life-saving anticancer dosage forms.

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