Analytical Techniques for Atorvastatin: Advances in Chromatography, Spectroscopy, and Hyphenated Methods

Atorvastatin calcium is a synthetic lipid-lowering agent belonging to the class of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, commonly referred to as statins [1–3]. Chemically, it is designated as (3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-(propan-2-yl)pyrrol-1-yl]-3,5-dihydroxyheptanoic acid, calcium salt (2:1) (Figure 1). The molecular formula of the calcium salt is (C??H??FN?O?)?Ca, and it has a molecular weight of approximately 1209.4 g/mol [1,2]. Atorvastatin acts by competitively inhibiting HMG-CoA reductase, a key enzyme involved in the mevalonate pathway of cholesterol biosynthesis in the liver. This inhibition leads to upregulation of hepatic LDL receptors, resulting in increased clearance of low-density lipoprotein cholesterol (LDL-C) from the circulation. In addition to lowering total cholesterol, LDL-C, and triglycerides, atorvastatin modestly increases high-density lipoprotein cholesterol (HDL-C), making it one of the most widely prescribed statins globally [3].

Figure 1: Chemical structure of Atorvastatin calcium

Atorvastatin is approved by several regulatory agencies, including the U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA), and Central Drugs Standard Control Organization (CDSCO), for the management of dyslipidemia, hypercholesterolemia, and prevention of cardiovascular events. It is indicated for both primary and secondary prevention of atherosclerotic cardiovascular disease (ASCVD), particularly in patients with risk factors such as diabetes, hypertension, or existing coronary artery disease [1–3]. Since its patent expiration, numerous generic formulations of atorvastatin have entered the global market. Consequently, regulatory authorities emphasize the need for stringent quality control measures during manufacturing, distribution, and post-marketing surveillance. According to USP monograph and ICH guidelines (such as Q3A/B for impurities and Q1A for stability), robust analytical methods must be employed for the accurate quantification of atorvastatin and its related substances in bulk drug substances and finished dosage forms. Given atorvastatin’s widespread therapeutic use, extensive analytical characterization is crucial to ensure the identity, purity, potency, and stability of both active pharmaceutical ingredient (API) and dosage forms. Furthermore, due to its susceptibility to oxidative and hydrolytic degradation, atorvastatin requires the application of stability-indicating methods compliant with ICH Q1A(R2) guidelines. Various analytical techniques, including UV-visible spectroscopy, FTIR, HPLC, UPLC, GC, LC-MS/MS, and other hyphenated methods, have been employed for its qualitative and quantitative estimation. The need for selective, sensitive, and reproducible methods is paramount, particularly in the context of bioequivalence studies, pharmacokinetic evaluations, forced degradation studies, and formulation development. Moreover, the growing emphasis on green and sustainable analytical chemistry further necessitates the development of eco-friendly, cost-effective, and regulatory-compliant methods for routine quality control of atorvastatin. Regulatory requirements play a vital role in ensuring the accuracy, reliability, and consistency of analytical methods for atorvastatin. As per ICH Q2(R2) and USP <1225>, all analytical procedures used in quality control must be validated for parameters such as accuracy, precision, specificity, linearity, detection limits, and robustness. Given atorvastatin's susceptibility to degradation, ICH Q1A(R2) and Q1B recommend stress testing under acidic, basic, oxidative, thermal, and photolytic conditions to develop robust stability-indicating methods [4–7]. The USP monograph for atorvastatin calcium outlines compendial tests including HPLC assay, impurity profiling, and dissolution testing, ensuring consistent quality across production batches. In pharmacokinetic studies, regulatory guidelines such as FDA bioanalytical method validation guidance and ICH M10 require validated LC-MS/MS methods that demonstrate selectivity, sensitivity, accuracy, and stability in biological matrices. With the growing global emphasis on environmental sustainability, the field of analytical chemistry has witnessed a transformative shift toward greener practices. Traditional analytical methods, though effective in precision and sensitivity, often involve the use of toxic solvents, generate substantial waste, and consume significant energy—contributing to environmental degradation and occupational hazards. As a result, assessing and improving the greenness of analytical procedures has become a crucial scientific and ethical responsibility [8–11]. Two widely accepted tools for evaluating the environmental impact of analytical methods are the Analytical Eco-Scale and the AGREE (Analytical GREEnness) metric [12,13]. These approaches serve as quantitative frameworks to systematically assess various aspects of analytical methods, including solvent toxicity, reagent consumption, waste generation, and energy usage. The Analytical Eco-Scale assigns penalty points based on hazard levels and resource consumption, offering a score out of 100 where higher values reflect more sustainable methods. On the other hand, AGREE is based on the 12 principles of Green Analytical Chemistry, generating a visual and numerical score between 0 and 1, thus providing a more holistic view of sustainability across the entire method lifecycle (Figure 2). The significance of these tools extends beyond the laboratory. By promoting cleaner, safer, and resource-efficient techniques, they align analytical science with the United Nations Sustainable Development Goals (SDGs), especially those related to health, clean water, and climate action. Adoption of greener methodologies not only enhances environmental protection but also fosters innovation, cost-effectiveness, and compliance with regulatory standards.

Figure 2: Analytical Eco-Scale and the AGREE (Analytical GREEnness) metric approaches serve as quantitative frameworks

Adherence to regulatory standards is vital for the successful approval of new drug applications, generic formulations, and for ensuring global compliance with pharmacopoeial requirements. Such compliance guarantees that atorvastatin-containing products remain safe, effective, and therapeutically reliable throughout their intended shelf life. This review provides a critical evaluation of recent advancements (2020–2025) in spectroscopic, chromatographic, and hyphenated analytical techniques employed for the estimation of atorvastatin across bulk materials, pharmaceutical dosage forms, and biological matrices. Special emphasis is placed on method development, validation processes, and alignment with current regulatory expectations. Additionally, this review highlights the importance of green analytical chemistry by examining the conceptual foundation, practical application, and societal impact of two prominent greenness assessment tools: the Analytical Eco-Scale and AGREE metric. Through the evaluation of published analytical methodologies using these frameworks, the review identifies current trends, potential challenges, and future opportunities in fostering environmentally sustainable practices within analytical sciences.

Physicochemical and Pharmacokinetic Profile of Atorvastatin

Chemical Structure and Properties

Atorvastatin calcium is a white to off-white crystalline powder, structurally classified as a pyrrole-based statin. The drug exists as a calcium salt with the empirical formula (C??H??FN?O?)?Ca·3H?O and a molecular weight of approximately 1209.4 g/mol. Its IUPAC name is (3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-(propan-2-yl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoic acid calcium salt (2:1) [1–3,14,15]. The molecule consists of a heptanoic acid side chain with two chiral centers at the 3 and 5 positions, both of which are in the (R)-configuration, essential for optimal inhibition of HMG-CoA reductase. It contains lipophilic aromatic rings, a fluorophenyl moiety, and multiple polar functional groups, contributing to its amphiphilic character. This structural diversity necessitates careful analytical considerations during method development [1–3,14,15].

Solubility, Stability, and Ionization Characteristics

Atorvastatin calcium is practically insoluble in aqueous media, particularly at low pH, but shows improved solubility in ethanol, methanol, and acetonitrile. The aqueous solubility is strongly pH-dependent, attributed to the presence of both carboxylic acid and hydroxyl functional groups. It exhibits a pKa of approximately 4.46 (carboxylic acid) and 9.37 (hydroxyl), making it weakly acidic in nature [1–3,14,15]. The compound is sensitive to acidic, alkaline, oxidative, and photolytic degradation. Notably, atorvastatin undergoes lactonization, a reversible transformation to its inactive lactone form, particularly under acidic conditions. This characteristic imposes challenges in developing stability-indicating analytical methods. Stability studies must account for these transformations during storage, handling, and method validation as per ICH Q1A(R2) guidelines.

Pharmacokinetics and Metabolic Profile

Atorvastatin is administered orally and demonstrates moderate absorption, with a bioavailability of approximately 12% due to extensive first-pass metabolism. Peak plasma concentrations are typically achieved within 1–2 hours (T_max) post-administration. The drug is highly protein-bound (~98%) and exhibits a large volume of distribution (~381 L), indicating extensive tissue penetration. Atorvastatin is metabolized primarily in the liver via the cytochrome P450 3A4 (CYP3A4) pathway to produce active ortho- and para-hydroxylated metabolites, which contribute significantly to its pharmacological activity. Its elimination follows biphasic kinetics, with a terminal half-life of approximately 14 hours for atorvastatin and 20–30 hours for active metabolites. Less than 2% of the administered dose is recovered unchanged in urine, with the majority excreted via bile following hepatic metabolism [1–3,14,15].

Due to its complex metabolic profile and presence of multiple active metabolites, sensitive and selective bioanalytical methods such as LC-MS/MS are required for accurate quantification in pharmacokinetic and bioequivalence studies, in accordance with FDA and EMA bioanalytical method validation guidelines.

Analytical method for Atorvastatin

UV-Visible Spectrophotometric Methods for the Estimation of Atorvastatin:

UV-Visible spectrophotometry remains a cornerstone analytical technique for the quantification of atorvastatin, both as a single agent and in combination with other drugs, due to its simplicity, cost-effectiveness, and adaptability for routine pharmaceutical analysis (Table 1). Recent advances have focused on improving sensitivity, selectivity, and robustness, as well as expanding the method’s applicability to complex formulations and biological matrices.

HPLC Methods for Atorvastatin Analysis

High-Performance Liquid Chromatography (HPLC) remains the gold standard analytical technique for the quantification and stability assessment of atorvastatin, a widely prescribed statin for hyperlipidemia and cardiovascular risk management. A comprehensive review of HPLC methods for atorvastatin is essential to consolidate advances in method development, including sensitivity, specificity, and robustness, especially in the context of complex pharmaceutical formulations and combination therapies (Table 2). Stability-indicating HPLC methods play a critical role in ensuring drug quality by effectively separating atorvastatin from its degradation products, thus supporting regulatory compliance with ICH guidelines (Q1A(R2), Q2(R1)). Furthermore, validated HPLC methods underpin bioanalytical studies for pharmacokinetic profiling and therapeutic drug monitoring. Reviewing these methods facilitates identification of gaps, optimization of chromatographic conditions, and integration of green chemistry principles, ultimately contributing to enhanced quality control, regulatory submissions, and patient safety.

HPTLC methods for atorvastatin:

High-Performance Thin-Layer Chromatography (HPTLC) is a valuable analytical tool for the estimation of atorvastatin due to its cost-effectiveness, rapid execution, and suitability for routine analysis. Reviewing HPTLC methods for atorvastatin is crucial to ensure regulatory compliance and quality assurance, particularly under ICH Q2(R2) guidelines. Many of these methods are stability-indicating and capable of identifying degradation products formed under stress conditions such as acid, alkali, oxidative, thermal, and photolytic environments. Such reviews provide insights into atorvastatin’s degradation behavior, supporting formulation development and shelf-life determination. HPTLC also aligns with green analytical chemistry (GAC) principles due to reduced solvent use and waste generation. Numerous studies report its successful application for the simultaneous quantification of atorvastatin with other drugs in fixed-dose combinations. The method’s applicability to polypharmacy scenarios makes it ideal for routine clinical and manufacturing settings. Recognized by global pharmacopeias, HPTLC is also effective for fingerprinting, impurity profiling, and IVIVC studies. A critical review can uncover literature gaps, such as underreported matrix applications or unexplored combinations, which may drive novel method development. Overall, systematic evaluation of HPTLC methods ensures reliable, eco-friendly, and regulatory-aligned analysis of atorvastatin in diverse pharmaceutical matrices (Table 3).

Hyphenated analytical techniques such as LC-MS and LC-MS/MS have become essential for the precise estimation of atorvastatin due to their superior sensitivity and specificity. These methods provide reliable qualitative and quantitative data even in complex biological and pharmaceutical matrices. Atorvastatin, administered at low doses and extensively metabolized into active hydroxy derivatives, requires highly sensitive techniques capable of detecting nanogram levels. LC-MS/MS allows simultaneous quantification of atorvastatin and its metabolites, offering comprehensive pharmacokinetic and pharmacodynamic profiles. Regulatory agencies, including the USFDA and EMA, endorse these techniques for bioanalytical method validation, ensuring compliance with ICH M10 and FDA guidance. Hyphenated methods are crucial for identifying unknown degradation products in stability-indicating studies under ICH-recommended stress conditions. They also enable effective therapeutic drug monitoring and evaluation of drug–drug interactions, especially given atorvastatin’s metabolism via CYP3A4. These methods support bioequivalence and toxicokinetic studies in clinical trials, facilitating accurate dose optimization and safety assessment. Additionally, their high reproducibility and selectivity make them indispensable for translational research. Overall, hyphenated techniques represent a gold standard for atorvastatin analysis in both research and regulatory contexts (Table 4).

The assessment of analytical methods using the Analytical Eco?Scale reveals a strong commitment to green chemistry principles across all 19 evaluated studies. With scores ranging from 80 to 92, every method qualifies as an “excellent green analysis,” as per the Eco?Scale interpretation criteria, where a score above 75 reflects environmentally favorable practices. The highest scorers—Tomikj?M (2024), Mohamed?AR (2025), Kasem?H (2025), and Akabari?AH (2023)—demonstrated outstanding greenness (scores ≥ 90), likely due to their use of non-toxic solvents, reduced reagent volumes, low energy consumption, and minimal hazardous waste generation. These methods set a benchmark for environmentally sustainable analytical practices. In the mid-range group, methods scoring between 84 and 86, such as those reported by El?Awady?MI (2023), Patel?S (2022), and Sukumar?V (2023), also showcased sound environmental design, though there may be room for improvement in terms of solvent selection or process efficiency. The relatively lower yet still excellent scores, observed in methods like those by Abu?Reid?IO (2021), Bagade?OM (2023), and Thomas?T (2021), which ranged between 80 and 83, may indicate the use of more hazardous solvents, higher reagent consumption, or greater energy requirements. Nevertheless, even these methods reflect a conscious effort toward greener practices. A notable trend is the higher Eco?Scale scores in more recent publications, highlighting the increasing awareness and adoption of green analytical chemistry over time. While AGREE scores were also uniformly high, the Eco?Scale provided more sensitivity in distinguishing subtle differences in environmental performance. Overall, this comparison underscores the positive trajectory toward environmentally responsible method development in pharmaceutical analysis (Figure 3). Continuous efforts such as adopting greener solvents, miniaturizing procedures, and optimizing energy usage can further enhance the sustainability of these analytical techniques.

Figure 3: Agree and Eco analytical scale score of analytical method for atorvastatin