Analytical Techniques for Monitoring Atorvastatin in Pharmaceuticals: A Detailed Review

Atorvastatin, a synthetic lipid-lowering agent from the statin class, is one of the most widely prescribed medications for managing hyperlipidemia and reducing cardiovascular risk. ^[1,5] Known for its high potency and efficacy, it plays a critical role in lowering low-density lipoprotein cholesterol (LDL-C) and preventing atherosclerotic progression.^[2] coronary artery disease and the progression of atherosclerotic lesions are closely linked to hyperlipidemia, which serves as a significant risk factor. Effective management of hyperlipidemia is crucial for preventing the development of atherosclerotic plaque, promoting its regression, and reducing the likelihood of acute coronary events in individuals with established coronary or peripheral vascular disease. ^[3] This management typically involves a combination of dietary modifications and lipid-lowering medications. Statins, among the most commonly prescribed drugs for both primary and secondary prevention of hyperlipidemia, play a central role in this therapeutic approach. ^[2] These medications lower plasma levels of cholesterol-rich lipoproteins and significantly reduce the risk of coronary heart disease. The pharmacological action of statins is attributed to their inhibition of the enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase), which is critical in the early stages of cholesterol biosynthesis. ^[4] By preventing the conversion of HMG-CoA to mevalonic acid, statins effectively interrupt the cholesterol production pathway. ^[5] Atorvastatin, a synthetic member of the statin class, is distinguished by its high potency and favorable pharmacokinetic profile. It has become one of the most widely used statins due to its ability to achieve significant reductions in low-density lipoprotein cholesterol (LDL-C) and its proven efficacy in reducing cardiovascular events. Additionally, atorvastatin has demonstrated pleiotropic effects, including anti-inflammatory and plaque-stabilizing properties, which further contribute to its therapeutic benefits. ^[3] The U.S. Food and Drug Administration authorized atorvastatin in 2003, marking the introduction of new synthetic compounds of the statin class in 1996. Atorvastatin was the world's top-selling medication in 2002. For the population to receive high-quality medications, quality control of this pharmaceutical product is therefore essential. As a result, this article reviewed the analytical techniques currently used for atorvastatin evaluation in the literature and official compendia. ^[6]

Structure of Atorvastatin:

Fig. 1: Atorvastatin Structure

Atorvastatin, a lipid-regulating drug (Figure 1), presents chemical name [(3R, 5R)-7-[2-(4-fluorophenyl)-3-phenyl4-(phenylcarbamoyl)-5-(propan-2-yl)-1H-pyrrol-1-yl]-3, 5-dihydroxyheptanoic acid, molecular formula C₃₃H₃₅FN₂O₅, and molecular weight 558.65 g/mol. Dr. Bruce Roth synthesized atorvastatin for the first time in 1985, and the FDA authorized it in 1996. With a 3, 5-dihydroxypentanoyl side chain that is identical to that of its parent molecule and an achiral heterocyclic core unit, it is a penta substituted pyrrole. Atorvastatin does not need to be activated, in contrast to other statin group members, because it is an active substance. ^[7]

Atorvastatin Calcium:

The marketed form is atorvastatin calcium, the trihydrate of calcium salt (2+1). It has molecular formula C₆₆H₆₈CaF₂N₄O₁₀, molecular weight 1209.41 g/mol, and white crystalline powder characteristics. Atorvastatin calcium is very soluble in acetonitrile, distilled water, and phosphate buffer, easily soluble in methanol, slightly soluble in ethanol, and insoluble in solutions of pH ≤4. ^[7] Atorvastatin calcium is marketed in the form of tablets. When used orally, the usual dosage ranges from 10 to 80 mg per day, and it is quickly absorbed. It is employed to prevent cardiovascular events. A medication used to lower the amount of cholesterol in the blood and decrease the chance of having a heart attack or stroke. ^[15]

Mechanism of Action:

Atorvastatin calcium reduces the amount of total cholesterol in the blood, decreasing levels of harmful fractions [low density lipoprotein (LDL)-C, apo-B, very low density lipoprotein (VLDL)-C, triglycerides] and increasing blood levels of beneficial cholesterol. Atorvastatin calcium inhibits the production of cholesterol by the liver and increases the absorption and destruction of harmful fractions (LDL) of cholesterol around 40–60 %. ^[15]

Pharmacokinetic Profile of Atorvastatin:

Absorption: When taken orally, atorvastatin is quickly absorbed. After dosage, the maximal plasma concentration (C_max) is reached in one to two hours. Because the liver is its main site of action and undergoes considerable first-pass metabolism, the absolute bioavailability is about 12%. Its strong hepatic absorption maintains its therapeutic efficacy despite its comparatively limited bioavailability. Its absorption rate may be affected by variables including pH, stomach motility, and co-administration with specific drugs (such proton pump inhibitors). ^[8]

Distribution: Atorvastatin is highly protein-bound (≥98%), primarily to albumin, which helps in its systemic distribution and plasma stability. It has a volume of distribution (V_d) of about 381 liters, indicating extensive tissue distribution, including into the liver and vascular walls, where it exerts its lipid-lowering effects. Minimal penetration across the blood-brain barrier has been observed, reducing potential central nervous system side effects. ^[9]

Metabolism: Atorvastatin undergoes extensive metabolism in the liver via the cytochrome P450 3A4 (CYP3A4) enzyme. The primary active metabolites are ortho-hydroxy and para-hydroxy derivatives, along with beta-oxidation products, which contribute to its pharmacological activity. These active metabolites account for a significant part of its lipid-lowering effect. Genetic polymorphisms in CYP3A4 or co-administration of CYP3A4 inhibitors (e.g., ketoconazole, grapefruit juice) or inducers (e.g., rifampin, St. John’s wort) may significantly alter its metabolism and therapeutic outcomes. ^[10]

Elimination: Atorvastatin is primarily eliminated in the bile after hepatic metabolism, with minimal renal excretion (<2% of the administered dose). It does not undergo significant enterohepatic recirculation, which helps maintain predictable pharmacokinetics. Atorvastatin's pharmacodynamic activity half-life is longer (~20-30 hours), allowing for once-daily treatment, despite its elimination half-life of about 14 hours due to the persistence of active metabolites. Dosage adjustments may be necessary if liver impairment (such as in cirrhosis patients) results in decreased clearance and a longer half-life. ^[11]

Food Effect: Food reduces the rate of atorvastatin absorption (decreases C_max by ~25%), but it does not significantly affect the overall extent of absorption (AUC), making it suitable for dosing with or without food. However, co-administration with certain high-fat meals may delay its absorption. It is generally recommended to administer atorvastatin at a consistent time each day, preferably in the evening, to align with the body’s natural diurnal cholesterol synthesis peak. ^[15]

Pharmacodynamic Profile of Atorvastatin:

Atorvastatin, a statin medication, primarily works by inhibiting HMG-CoA reductase, an enzyme crucial for cholesterol synthesis in the liver. This inhibition lowers intracellular cholesterol levels, triggering an upregulation of LDL receptors on liver cells, which in turn enhances the clearance of low-density lipoprotein cholesterol (LDL-C) from the bloodstream.^{[13, 14]} This leads to significant reductions in LDL-C, moderate increases in high-density lipoprotein cholesterol (HDL-C), and substantial decreases in triglycerides (TG). The lipid-lowering effects become noticeable within two weeks of starting treatment and peak after 4-6 weeks, persisting as long as the medication is taken.^[12] Beyond its cholesterol-lowering effects, atorvastatin exhibits additional benefits, such as anti-inflammatory and antioxidant properties. These effects help improve endothelial function, reduce vascular inflammation, and stabilize atherosclerotic plaques, contributing to its cardiovascular protective role.^[16] It is also effective in reducing C-reactive protein (CRP) levels, a marker of systemic inflammation. Clinically, atorvastatin is used to manage hypercholesterolemia, mixed dyslipidemia, and familial hypercholesterolemia, and for the prevention of cardiovascular events like myocardial infarction and stroke. Pharmacodynamically, atorvastatin demonstrates dose-dependent effects, with higher doses yielding more pronounced lipid-lowering benefits.^[14] The medication is metabolized by the CYP3A4 enzyme, so interactions with drugs or substances affecting this pathway, such as strong CYP3A4 inhibitors or grapefruit juice, can increase its plasma concentrations and the risk of adverse effects like myopathy.^[16] While generally well-tolerated, atorvastatin can cause side effects such as muscle pain, gastrointestinal disturbances, and elevated liver enzymes, with rare but serious risks including rhabdomyolysis and hepatotoxicity. It is contraindicated in active liver disease but does not require dose adjustments in renal impairment due to its hepatic elimination.^[17] Pharmacodynamics of atorvastatin highlight its multifaceted role in cardiovascular risk reduction, leveraging both lipid-lowering and pleiotropic effects to provide significant therapeutic benefits.

Analytical Account on Atorvastatin:

The following techniques were used in the literature to assess atorvastatin: HPLC, HPLC coupled to MS (HPLC-MS), TLC, UV-Vis, CE, HPTLC, Raman, X-ray diffraction, namely, matrix-assisted laser desorption ionization, dissolution, voltammetry, spectrofluorimetric, IR, electrokinetic capillary chromatography, ultra-performance LC (UPLC), UPLC coupled to MS. The majority of them, or about 70%, use HPLC, HPLC-MS, and UV techniques. The most popular technique for analyzing biological samples is the HPLC-MS method, while the most popular technique for analyzing pharmaceutical samples is the HPLC method. The majority of the studies discuss pharmaceutical samples of atorvastatin, including tablets, bulk, bulk impurities, nanoemulsions and nanocrystals. Papers concerning biological samples (serum and plasma), are included. The most researched samples are atorvastatin tablets and atorvastatin in plasma.^[6]

UV-Visible Spectroscopy Method

Ashok H. Akabari et al. used HPLC and UV Spectrophotometric Methods to Estimate Fimasartan potassium trihydrate and Atorvastatin calcium Simultaneously with Greenness Assessment. Spectrophotometric measurements were conducted using a double-beam UV/VIS spectrophotometer, with a spectral bandwidth of 1 nm and wavelength accuracy of ± 0.5 nm. The analysis was performed using UV Probe software, with absorbance readings taken in 1 cm quartz cells. Method I employs Vierordt’s simultaneous equation, while Method II utilizes First Order Derivative Spectrophotometry. In Method I (Vierordt’s method), the zero-order UV absorption spectra revealed overlapping absorption bands for the two drugs within the range of 200–320 nm, complicating their quantification via conventional UV spectrophotometry without correcting for spectral overlap. Method II (First Order Derivative Spectrophotometry) was implemented to resolve this overlap. Various scaling factors and Δλ values were tested to optimize the position and amplitude of the zero-crossing points. The first derivative spectra showed that atorvastatin exhibited a peak at 261.60 nm.^[18] Abu Reid et al. described Simple spectrophotometric methods for the determination of amlodipine and atorvastatin in bulk and tablets. The aim of this study was to utilize multiwavelength regression analysis and the absorbance factor method as simple, cost-effective, and reliable UV-spectrophotometric techniques, offering an alternative to expensive separation-based or sophisticated methods requiring specialized software. All analytical measurements were performed using a Shimadzu UV-1800 double-beam UV/Vis spectrophotometer fitted with two 1 cm quartz cells. Atorvastatin's UV absorbance spectra (23 µg/mL) were captured between 200 and 400 nm in wavelength, with the diluent (50% v/v aqueous methanol) serving as the blank. The diluent was prepared by combining equal volumes of methanol and distilled water. Atorvastatin exhibited maximum absorbance at 244 nm and no absorbance at 365 nm. The absorbance of atorvastatin at 244 nm was quantified using the absorbance factor method. For the multilinear regression analysis, absorbance measurements were made at 10 nm intervals between 230–260 nm. Calibration curves for each method were linear within the concentration range, with R² values exceeding 0.99.^[19] Elsaman T. et al. developed and validated a UV-spectrophotometric method for determining atorvastatin calcium using Sodium Citrate as Hydrotropic Agent. UV absorbance was measured at 241 nm using sodium citrate (0.01 M) as a hydrotropic agent. In the concentration range of 2–20 µg/mL, the method's R² value was 0.999, in accordance with Beer's law. The quantification and detection limits were found to be 1.9 µg/mL and 0.64 µg/mL, respectively. RSD% values below 2% were found at all levels in precision studies. The standard addition method was used to verify the procedure's accuracy, and the percentage recoveries (n = 3) ranged from 100% to 100.43%. Three atorvastatin tablet brands, each labeled to contain 20 mg of atorvastatin, were successfully analyzed using the approach. The percentage drug content was found to be 101.42 ± 1.56%, 99.04 ± 0.33%, and 97.71 ± 0.98%, respectively.^[20] Yilmaz Bilal et al. described UV and First Derivative Spectrophotometric Methods for the Estimation of Atorvastatin in Pharmaceutical Preparations. Optimal results were achieved by measuring the wavelength range of 220–320 nm with high smoothing (Δλ = 21.0 nm) for both UV spectrophotometry and first-order derivative (¹D) spectrophotometry. In the UV spectra of atorvastatin, a maximum peak was observed at 247 nm. A maximum peak and a minimum peak were observed at 237 and 261 nm, respectively, in the first-order derivative spectra. Six-level calibration series, with six replicate analyses at each concentration level, were used for quantification. The first derivative of absorbance (dA/dλ) and absorbance (A) were plotted against atorvastatin concentrations to create standard calibration curves. Both methods demonstrated excellent linearity in the 5–20 µg/mL concentration range. The average percent recoveries were 100.3% for the UV method and 99.8% for the ¹D method, confirming the high accuracy of both analytical approaches.^[21] Jayasundara et al. described Method Development, Validation, and Concentration Determination of Metformin Hydrochloride and Atorvastatin Calcium Using UV-Visible Spectrophotometry. The UV-Vis spectra of atorvastatin calcium tablets showed a maximum absorbance (λmax) at 233 nm, which was used for subsequent analyses. A double-beam UV spectrophotometer was employed to measure the absorbance of all prepared samples. The concentration range of 5–15 ppm was used to generate a five-point calibration curve, demonstrating linearity with an R² value of 0.998 for atorvastatin calcium. The spike recovery method was used to assess the approach's accuracy, and mean recoveries ranging from 90.10% to 102.90% were obtained. The developed method determined the actual concentration of active ingredients in atorvastatin calcium tablets to range from 9.00 to 9.88 mg per 10 mg labeled dose.^[22] Hany W. Darwish et al. described three different methods for determination of binary mixture of Amlodipine and Atorvastatin using dual wavelength spectrophotometry. The new Ratio Difference method is the first approach, followed by the Bivariate method and the Absorbance Ratio method. SHIMADZU dual beam UV–visible spectrophotometer model UV-1650 PC connected to IBM compatible and a HP1020 laserjet printer. The bundled software, UV-Probe personal spectroscopy software version 2.21 is used. The spectral band is 2 nm and scanning speed is 2800 nm/min with 0.1 nm interval.^[23] Ashour S et al. described New Kinetic Spectrophotometric Method for Determination of Atorvastatin in Pure and Pharmaceutical Dosage Forms. Utilizing an oxidative coupling reaction with Ce(IV) in an acidic medium, the approach forms a colorful product with a maximum absorbance (λmax) at 566 nm by combining atorvastatin calcium with 3-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH). A UV-VIS spectrophotometer fitted with 1 cm quartz cells was used to measure absorbance under the following operating conditions: a scan speed of 400 nm/min, a scan range of 400–800 nm, and a slit width of 2 nm. By tracking the rise in absorbance at 566 nm over time, the process was observed spectrophotometrically. The initial rate and fixed time methods were used to generate calibration curves, and both procedures had linearity ranges of 2.0–20.0 µg/mL. For both the initial rate and fixed time approaches, the limits of detection were 0.093 µg/mL and 0.064 µg/mL, respectively. 3.36 × 10? L/mol cm was found to be the method's molar absorptivity.^[24]

Sagar Patel et al. described validated chromatographic methods for simultaneous measurement of atorvastatin in synthetic combination. A 60F254 pre-coated silica gel aluminum plate measuring 10 cm x 10 cm and 0.2 mm thick was used as the stationary phase. The mobile phase was composed of glacial acetic acid, methanol, dichloromethane, and ethyl acetate in a volumetric ratio of 6:2:2:0.1 (v/v/v/v). 8 mm bands were placed 9 mm from the plate's lowest edge. A Camag TLC scanner operated by Camag winCATS software (Version 1.4.8) was used for densitometric analysis. At a wavelength of 221 nm, atorvastatin was detected, and the Rf value was 0.70 ± 0.02. Using linear regression analysis, a strong linear association (R² = 0.9993) was found between the peak region and the 800–4800 ng/band concentration range.^[25] Noha N. Atia et al. described Novel sublingual tablets of Atorvastatin calcium/Trimetazidine hydrochloride combination; HPTLC quantification, in vitro formulation and characterization. The stationary phase consisted of aluminum plates pre-coated with silica gel G 60F254, measuring 20 cm × 20 cm with a layer thickness of 0.20 mm. Chloroform, methanol, and glacial acetic acid were combined in a volumetric ratio of 68:11.2:0.8 (v/v/v) to create the mobile phase. Sample application was performed using a Camag HPTLC system equipped with a Linomat-5 automatic sample applicator and a 100 µL Camag syringe, applying samples as 4 mm bands. After chromatographic development, a dual-wavelength TLC scanner was used to scan the plates in absorbance mode at a wavelength of 246 nm. Calibration curves exhibited excellent linearity over the concentration range of 0.05–1.0 µg/band, with an R² value exceeding 0.9994.^[26] Ilango et al. described a development and subsequent validation of stability indicating HPTLC methods for estimation of Atorvastatin in formulation. In the HPTLC method, the drug was separated chromatographically using silica gel 60F254 plates (10.0 × 10.0 cm with a layer thickness of 250 µm) as the stationary phase. In a volumetric ratio of 5:1:1:0.3 (v/v), toluene, methanol, ethyl acetate, and acetic acid made up the mobile phase. Sample application was performed as 8 mm bands using a 100 µL sample syringe and Linomat 5 applicator. A TLC scanner was used to perform densitometric scanning at 279 nm in absorbance/reflectance mode with an Rf value of 0.63 ± 0.01. Linear regression study for atorvastatin revealed linearity in the 10–60 ng/band concentration range. The limits of quantification and detection for this approach were found to be 6.22 ng/band and 2.13 ng/band, respectively. Recovery studies conducted at three different concentrations in triplicate revealed satisfactory recoveries of atorvastatin, ranging from 97.86% to 98.81%.^[27] Panchal et al. described Simultaneous Analysis of Atorvastatin Calcium and Losartan Potassium in Tablet Dosage Forms by RP-HPLC and HPTLC. A mobile phase comprising methanol, carbon tetrachloride, ethyl acetate, and glacial acetic acid in a volumetric ratio of 8:63.6:28:0.4 (v/v/v/v) was used to conduct separation on silica gel 60F254 plates by HPTLC. Atorvastatin calcium had a retardation factor (Rf) of roughly 0.45. Using ultraviolet (UV) detection at 238 nm and having a 50–500 ng/band linear concentration range, quantification was done. The method exhibited a mean recovery of 100.59% ± 0.47 for atorvastatin calcium.^[28] Ma?lanka, Anna et al. discussed the simultaneous TLC determination of atorvastatin, enalapril, hydrochlorothiazide, and acetylsalicylic acid in a polypill-based quaternary mixture. TLC plates coated with silica gel 60 F₂₅₄, which has a fluorescent indicator as the stationary phase, were used for chromatographic separation. A mixture of n-hexane, ethyl acetate, methanol, water, and acetic acid in the volumetric ratio 8.4:8:3:0.4:0.2 (v/v/v/v/v) made up the mobile phase. The wavelength used for densitometric measurements was 265 nm. Both qualitative and quantitative assessment were made possible by the chromatograms' distinct separation of the peaks that corresponded to the compounds under analysis. The technique showed great sensitivity and specificity for the target components. The range of the recovery rate was 97.02% to 101.34%. The linearity of the process was shown for atorvastatin at concentrations between 0.100 and 1.000 µg/spot. Furthermore, the method's relative standard deviation (RSD) values, which ranged from 0.10% to 2.26%, demonstrated acceptable precision.^[29]

Marija Tomikj et al. proposed a sustainable and "white" high-performance liquid chromatography (HPLC) method for the simultaneous quantification of amlodipine and atorvastatin in film-coated tablet formulations. Using a Zorbax SB-C8 column (150 × 4.6 mm, 5 µm) and a mobile phase made up of ethanol and 0.02 M sodium dihydrogen phosphate monohydrate, which had been adjusted to pH 3.0 using orthophosphoric acid in a 63:37% (v/v) ratio, the chromatographic separation was carried out. A 10 µL injection volume and a flow rate of 0.8 mL/min were maintained. Analytes were detected at a wavelength of 254 nm, and the column temperature was set at 40°C. Atorvastatin eluted at a retention time of roughly 3.3 minutes during the 5-minute chromatographic run. Five standard solutions generated within the concentration range of 0.05 to 0.15 mg/mL were used to evaluate the method's linearity.^[30] Ashok H. Akabari et al. described a method for the simultaneous estimation of Fimasartan potassium trihydrate and Atorvastatin calcium, incorporating greenness assessment, using both HPLC and UV spectrophotometric techniques. A Shim-Pack ODS C18 column (4.6 mm × 250 mm, 5 µm) was used as the stationary phase to accomplish chromatographic separation. A photodiode array detector tuned at 234 nm was used for detection. Acetonitrile and 0.01 M ammonium acetate buffer (pH adjusted to 3.7 with formic acid) were supplied in a 70:30 (v/v) ratio as the mobile phase, with a steady flow rate of 1 mL/min.  In the binary mixture's chromatogram, atorvastatin eluted in 8.79 ± 0.13 minutes with acceptable system suitability criteria. Excellent injection repeatability was confirmed by the relative standard deviation (RSD) of peak area response and retention time for six consecutive injections, which was 2.0%.  The theoretical plate number was more than 2000, showing sufficient column efficiency, while the tailing factor was less than 1.3, suggesting satisfactory peak symmetry.^[31] Maged, K. et al. described a Development and validation of an eco-friendly HPLC–UV method for determination of atorvastatin and vitamin D3 in pure form and pharmaceutical formulation. An Alliance 2695 HPLC system with a quaternary gradient pump, autosampler, column oven, and photodiode array (PDA) detector was used for the HPLC analysis. On a Symmetry C18 column (100 × 4.6 mm, 3.5 µm particle size), separation was performed using a gradient mobile phase made up of ethanol and 0.1% ortho-phosphoric acid (pH 2.16). The column temperature was kept at 40°C while the mobile phase was supplied at a flow rate of 1 mL/min. The retention time of atorvastatin was roughly 6.12 minutes. The atorvastatin calcium was detected at 246 nm using a 20 µL injection volume. With a correlation coefficient (r2) of 0.9998, the technique showed excellent linearity for atorvastatin concentrations between 5 and 40 µg/mL.^[32] Alruwaili, Nabil K. et al. described the use of analytical quality by design (AQbD) approach in the optimization of the high-performance liquid chromatography (RP-HPLC) method as a novel tool. A Box–Behnken statistical design with three factors and three levels was employed for the optimization and analysis of atorvastatin. Chromatographic separation was accomplished with a (250 mm × 4.6 mm, 2.2 μm) Acclaim 120 C18 column. Acetonitrile and water in a 50:50 (v/v) ratio made up the optimum mobile phase, which was supplied at a flow rate of 0.68 mL/min. The study was done at room temperature, and the detection was done at a UV wavelength of 235 nm. Atorvastatin was retained for 2.43 minutes at an injection volume of 30 μL. The technique demonstrated linearity with a strong correlation coefficient (R² = 0.999) over the concentration range of 5–30 μg/mL. The %RSD readings were less than 5%, demonstrating the method's accuracy and precision. It was determined that this Quality by Design (QbD)-optimized approach was appropriate for pharmacokinetic research and the measurement of atorvastatin in pharmaceutical formulations.^[33] Shulyak, N. et al. described a method for the determination of the most prescribed antilipemic drug, atorvastatin, together with its related substances. Chromatographic separation was carried out using a Shim-pack XR II C18 column (75 mm × 3 mm, 2.2 µm) with a mobile phase consisting of 0.05% v/v formic acid (pH adjusted to 4.0 with ammonium hydroxide) and acetonitrile in a single-step gradient elution mode. Atorvastatin had a retention duration of approximately 7.5 minutes, and the flow rate was fixed at 1.5 mL/min. Peak identity was confirmed through DAD UV spectral analysis at 244 nm and by spiking samples and standards with individual reference substances for each specified impurity. A notable advantage of this method is its ability to simultaneously quantify atorvastatin and its impurities in a single chromatographic run.^[34] Habib A.A. et al. developed an innovative Quality by Design (QbD) approach for the green micellar HPLC method for simultaneous determination of atorvastatin and amlodipine. A Box–Behnken design was used to investigate and optimize important parameters after a two-level fractional factorial design was used to screen different method parameters affecting chromatographic results. Using a mobile phase consisting of 0.17 M SDS solution (adjusted to pH 2.9 with diluted phosphoric acid) and 10% v/v n-butanol, chromatographic separation and quantification were carried out on an X-Bridge column (150 mm × 4.6 mm, 5 μm) at a flow rate of 1.5 mL/min. The injection volume was 50 μL, and the column temperature was kept at 45°C. Atorvastatin had a retention time of approximately 3.2 minutes. Fluorescence detection was carried out with excitation/emission wavelengths of 276/378 nm for the first five minutes, switching to 366/442 nm afterward. The method demonstrated excellent linearity in the range of 0.2–25 μg/mL, with a correlation coefficient (r²) of 0.9998.^[35] Piponski M et al. developed a fast, simple, and stability-indicating HPLC method for the analysis of atorvastatin and its related compounds in tablets. The method was designed to determine atorvastatin, its seven main specified impurities, and any unspecified impurities that may appear. Chromatographic separation and quantification were performed on an Octadecylsilyl C18 column (250 mm × 4.6 mm, 5 µm). The mobile phase consisted of a binary system composed of phosphate buffer and acetonitrile at pH 4.1, avoiding the use of tetrahydrofuran, ion-pair reagents, trifluoroacetic acid, or other high UV cut-off modifiers such as acetate, formate buffers, or amines. The chromatographic runs lasted between 25 and 40 minutes, employing a simple stepwise gradient elution. Detection was performed at 244 nm, with the analysis carried out at 30°C. ^[36] Alhazmi HA et al. developed a fast and validated reversed-phase HPLC method for the simultaneous determination of simvastatin, atorvastatin, telmisartan, and irbesartan in bulk drugs and tablet formulations. A Symmetry C18 column (75 mm × 4.6 mm, 3.5 µm) was used for the chromatographic separation, and the mobile phase was made up of 40:60 v/v acetonitrile and 10 mM ammonium acetate buffer (pH 4.0). For the first 3.5 minutes, the flow rate was kept at 1 mL/min. After that, it was raised to 2 mL/min until the analysis's conclusion at 7.5 minutes. The retention time of atorvastatin was almost 1.82 minutes. The UV wavelength used for detection was 220 nm. In the concentration range of 1–16 µg/mL, the technique showed high linearity (R² > 0.999). The results showed that the limits of quantification (LOQ) were 0.603–0.630 µg/mL and the limits of detection (LOD) were 0.189–0.190 µg/mL. All four medications were effectively quantified using this method in tablet dose forms, with recovery percentages falling within the range of 100 ± 2%.^[37] V. Sree Janardhanan et al. proposed a chemometric approach to optimize a chromatographic system for the simultaneous high-performance liquid chromatography (HPLC) analysis of rosuvastatin, telmisartan, ezetimibe, and atorvastatin in combination therapy for cardiovascular conditions. A Phenomenex C18 analytical column (150 mm × 4.6 mm, 5 µm) and a Phenomenex C18 guard cartridge (4 mm × 3 mm, 5 µm) were used for chromatographic separations. A solution of methanol (MeOH), acetonitrile (MeCN), and dipotassium hydrogen phosphate buffer (pH 3.0), with 10% phosphoric acid added for adjustment, made up the mobile phase. The material was analyzed using a 20 µL injection volume, and detection was performed at 239 nm. About 10 minutes were spent on the chromatographic run in total. Calibration curves, plotting the peak area ratios of atorvastatin to the internal standard against drug concentrations, were established within the range of 0.5–5 µg/mL.^[38] Jaiprakash N. et al. described the development and validation of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the determination of atorvastatin calcium and nicotinic acid in a combined tablet dosage form. An Agilent ZORBAX SB-C18 column (150 × 4.6 mm, 3.5 µm) was used for the analysis, and a mobile phase consisting of acetonitrile and distilled water (85:15) at pH 4.5 that had been adjusted with phosphoric acid was used. The flow rate was set at 1.0 mL/min, and the detection wavelength was 261 nm. It ran for 10 minutes in total. The retention time for atorvastatin calcium was 6.092 minutes. For atorvastatin calcium, the technique showed linearity across the 2–12 µg/mL concentration range. 99.03% of the atorvastatin calcium was recovered.^[39] Sahu, Prafulla Kumar et al. described the development and validation of a simultaneous reverse-phase high-performance liquid chromatography (RP-HPLC) method for the analysis of atorvastatin, ezetimibe, and fenofibrate. A reversed-phase C-18 column (25 mm × 4.6 mm, 5 µm) was used for the chromatographic separation, and the mobile phase was a 90:10 (v/v) mixture of acetonitrile and phosphate buffer (pH 3.3). The detection was done at 254 nm, and the flow rate was set at 1 mL/min. A 20 µL injection volume was employed, and the column was kept at ambient temperature. A % relative standard deviation (RSD) of less than 2% was obtained from precision and accuracy studies, and the method was found to be specific, simple, accurate, precise, sensitive, and validated. The run time was 9 minutes, and the retention time for atorvastatin was 3.155 minutes. The method demonstrated linearity for atorvastatin within the concentration range of 10–100 µg/mL (r² = 0.9985).^[40] Saroj Kumar Raul et al. described the development and validation of a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of atorvastatin and ezetimibe in pharmaceutical dosage forms. For chromatographic separation, a Hypersil BDS C18 column (250 mm × 4.6 mm, 5 µm) was used. Acetonitrile and phosphate buffer (pH 4.5) were mixed in a 35:65 (v/v) ratio and flowed at a rate of 1 mL/min to form the mobile phase. At 228 nm, a photodiode array detector was used for detection. 6 minutes were allotted for the run, and the sample injection volume was 10 µL. Atorvastatin was shown to have a retention duration of 2.36 minutes. Within the concentration range of 12.5–75 µg/mL, atorvastatin showed linearity. Atorvastatin's percentage recovery was 100.21%, demonstrating the method's high accuracy.^[41] Talele GS et al. described the development of a validated bioanalytical HPLC-UV method for the simultaneous estimation of amlodipine and atorvastatin in rat plasma. A 100 mm × 4.6 mm, 5 µm Thermo beta-basic C18 column was used in the procedure, along with a mobile phase that contained acetonitrile and dibasic phosphate buffer (pH 3.0) in a 55:45 ratio and flowed at a rate of 1 mL/min. A wavelength of 240 nm was used for ultraviolet detection. The retention time of atorvastatin was 12.1 minutes, and it was successfully separated under the chromatographic conditions. Using an unweighted calibration curve, the method demonstrated excellent linearity for both medications in rat plasma, with a correlation coefficient of 0.999 for the concentration range of 0.05 to 10.0 µg/mL. The protein precipitation approach yielded a mean recovery of nearly 92.8% for both medicines. For six replicate measurements, the accuracy at the lower limit of quantification fell within allowable bounds.^[42] Ilango et al. described the development and validation of a stability-indicating HPLC method for estimating atorvastatin in formulations. A Phenomenex Luna C18 column (5 µm × 25 cm × 4.6 mm) was used in the suggested RP-HPLC procedure. The mobile phase was made up of acetonitrile and 0.025 M ammonium acetate (38:52%, v/v) at pH 3.8, flowing at a rate of 1.0 mL/min. A PDA detector tuned at 281 nm was used for quantification, and atorvastatin showed a retention duration of 3.21 minutes. For atorvastatin, the technique produced linear calibration plots in the 3–18 µg/mL range. The results showed good sensitivity with limits of detection (LOD) and quantification (LOQ) of 1.21 µg/mL and 2.25 µg/mL, respectively. Both intraday and interday precision showed low relative standard deviation (RSD) values (<2.0%), indicating great precision. The recovery investigation confirmed the accuracy of the approach with an RSD ranging from 0.58 to 1.71%. Furthermore, atorvastatin and its main degradation products had a resolution of ≥2.0, demonstrating the method's robustness.^[43] Al-Akkam et al. developed a sensitive and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method for determining atorvastatin and rosuvastatin in rat plasma using a BDS Hypersil C18 column (250 mm × 4.6 mm, 5 µm). The pH was adjusted to 3.0 with trifluoroacetic acid, and isocratic elution was performed with methanol and water (68:32 and 63:37, v/v) at a 1 mL/min flow rate. A 100 µL plasma sample was injected, detected at 241 nm, and prepared via liquid-liquid extraction with methyl-tert-butyl ether following protein precipitation using ammonium acetate buffer. The retention times were 11.35 minutes for atorvastatin and 15.73 minutes for the internal standard. The calibration curve for atorvastatin was linear across 20–200 ng/mL, with an LLOD of 1.35 ng/mL, an LLOQ of 10.3 ng/mL, and an average recovery of 96.48%, demonstrating the method’s sensitivity and reliability for bioanalysis.^[44] Sher Muhammad et al. described a sensitive and rapid high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection, which was developed and validated for the determination of atorvastatin in human plasma. A C18 analytical column was used for the chromatographic separation, and the mobile phase was made up of 40:30:30 (v:v:v) methanol, acetonitrile, and sodium phosphate buffer (0.01 M, pH 4.5). At 247 nm, UV detection was carried out. With a limit of detection (LOD) of 7.82 ng/ml and a limit of quantification (LOQ) of 22.86 ng/ml, the technique demonstrated an average recovery of 98.7%. Over the atorvastatin concentration range of 5 to 160 ng/ml, the calibration curve was linear. Chromatographic studies of excipient mixtures verified the system's specificity and selectivity, demonstrating no interference at the atorvastatin retention time. A randomized, crossover bioequivalence study of two distinct atorvastatin tablet formulations—Lipirex, the generic version, and Lipitor, the brand leader—was conducted using the validated approach.^[45] Kumar P. et al. developed a stability-indicating RP-HPLC method for the simultaneous estimation of ezetimibe and atorvastatin in pharmaceutical formulations. Using a Zorbax SB C18 column (250 mm × 4.6 mm, 5 µm particle size) and a mobile phase made up of 0.02 M potassium dihydrogen phosphate, acetonitrile, and methanol in a 10:40:50 (v/v/v) ratio, chromatographic separation was accomplished. UV detection was used to detect at 236 nm, and the flow rate was set at 1.1 mL/min. A 20 µL injection volume was employed, and the column was kept at room temperature. Within 10 minutes, both analytes were well separated, showing minimal peak tailing and good resolution free from excipient interference. The retention time for atorvastatin was 9.1 minutes. For atorvastatin values between 5 and 60 µg/mL, the technique was linear, with a recovery range of 99–102% and a correlation coefficient of 0.9994.^[46] Yasar Shah et al. reported the simultaneous quantification of rosuvastatin and atorvastatin in human serum utilizing a reverse-phase high-performance liquid chromatography (RP-HPLC) method with UV detection. Atorvastatin was separated chromatographically using a Brownlee analytical C18 column (150 × 4.6 mm, 5 µm) and a mobile phase made up of methanol and water in a 68:32 (v/v) ratio that was brought to pH 3.0 using trifluoroacetic acid. A 20 µL injection volume, a 1.5 mL/min flow rate, and UV detection at 241 nm were all used in the procedure. At 25°C, the column oven was kept constant. A retention time of roughly 6.0 minutes was demonstrated by atorvastatin. With an average recovery of more than 98.0%, the atorvastatin calibration curves were linear over the concentration range of 3–384 ng/mL. For atorvastatin, the technique showed a lower limit of quantification (LLOQ) of 3.0 ng/mL and a lower limit of detection (LLOD) of 1.0 ng/mL. Analyte separation was completed in 7.0 minutes, and the %RSD values for intra-day and inter-day precision were less than 2.0%.^[47] Panchal et al. described a method for the simultaneous analysis of atorvastatin calcium and losartan potassium in tablet dosage forms using both RP-HPLC and HPTLC techniques. A Phenomenex Luna C18 column (250 mm x 4.6 mm, 5 μm) was used for separation in the RP-HPLC method. A mobile phase made up of 0.05 M potassium dihydrogen phosphate buffer (pH 5.4) and acetonitrile in a 45:55 (%, v/v) ratio was used. A constant flow rate of 1 mL/min was maintained. The retention period for atorvastatin calcium was roughly 7.56 minutes. At 238 nm, a photodiode-array (PDA) detector was used for quantification. With a mean recovery of 100.67 ± 0.58 for atorvastatin calcium, the method demonstrated linearity over the concentration range of 0.5–5 μg/mL, demonstrating good accuracy and precision for the analysis.^[48] Mohammadi A et al. developed a stability-indicating HPLC assay for the simultaneous determination of atorvastatin and amlodipine in commercial tablets. A Perfectsil Target ODS-3 column (5 μm, 250 mm × 4.6 mm i.d.) was used for the chromatographic separation, and the mobile phase was made up of acetonitrile and 0.025 M NaH₂PO₄ buffer at pH 4.5 in a 55:45 (v/v) ratio. UV detection was carried out at 237 nm, and the flow rate was fixed at 1 mL/min. The injection volume was 20 μL, and the column was kept at room temperature (25°C). With a corresponding %RSD value of 0.13% and a retention time of 9.5 minutes, atorvastatin demonstrated exceptional precision. It was demonstrated that the technique successfully separated amlodipine and atorvastatin from the excipients, related compounds, and degradation products that are present in tablet dosage forms. It was a dependable technique for regular quality control and stability testing of these medications, and it was also appropriate for evaluating samples from accelerated stability studies.^[49] Gholamreza Bahrami et al. developed a method for the determination of atorvastatin in human serum using reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection. A Shim-pack CLC-ODS analytical column (150 mm × 4.6 mm I.D., 5 µm particle size) was used to accomplish the separation. The mobile phase was made up of 33:67 (v/v) methanol and 0.05 M sodium phosphate buffer, which had been adjusted to pH 4.0 using orthophosphoric acid. The mobile phase was pumped at a rate of 2.5 mL/min while the column was kept at 62°C. Atorvastatin was eluted with a retention duration of 3.4 minutes under these circumstances. At 247 nm, the detection was carried out. With a lower limit of detection (LOD) of 1 ng/mL and a lower limit of quantification (LOQ) of 4 ng/mL, the technique demonstrated an average recovery of 95%. For atorvastatin in human serum, the calibration curve was linear in the concentration range of 4–256 µg/mL, which makes it appropriate for accurate atorvastatin quantification in biological samples.^[50]

Erokhina P.D. et al. developed and validated a mass spectrometric detection (MS/MS) technique in conjunction with high-performance liquid chromatography (HPLC) for the quantitative measurement of atorvastatin in the HepG2 cell line. A UCT Selectra C18 column (4.6 mm × 100 mm, 5 µm) and a Selectra C18 Guard Cartridges pre-column were used for the chromatographic analysis, which was carried out at a flow rate of 0.3 mL/min. The column was thermostated at 35°C, and a sample volume of 2 µL was injected. The total analysis time was 10 minutes. The makeup of the mobile phase was progressively changed, starting with a mix of formic acid solution and acetonitrile, switching to a higher concentration of acetonitrile, and then returning to the original ratio for improved separation. The retention time for atorvastatin was 4.53 minutes. The mass spectrometric detection was conducted in positive ionization mode with a spray voltage of 3500 V. Multiple reaction monitoring (MRM) was used with transitions at 559.30 m/z → 466.20 m/z and 559.30 m/z → 440.20 m/z. 17 V was the collision energy, and the source fragmentation was 0, with a gas pressure of 2 mTorr. This method provided a reliable and sensitive approach for determining atorvastatin concentrations in the HepG2 cell line, offering significant advantages in terms of specificity and sensitivity.^[51] Elawady et al. developed a method for the simultaneous determination of ezetimibe, atorvastatin, and simvastatin in combined tablets and plasma using quadrupole LC-MS following solid-phase extraction (SPE). Using a Zorbax Eclipse Plus C18 column (3.0 x 150 mm, 5 µm) and a mobile phase consisting of acetonitrile and 0.1% formic acid in water (65:35, v/v) at a flow rate of 0.5 mL/min, a sample injection volume of 20 µL, and a run time of 9.3 minutes, chromatographic separation was accomplished. The internal standard (IS) was diclofenac sodium, and the retention time for atorvastatin was 2.46 minutes. The atorvastatin-corresponding ions at m/z 559.3 were monitored using the positive selected ion monitoring (SIM) method. With intraday and interday precision displaying relative SD values of no more than 1.77% and 1.99%, respectively, the approach was validated in accordance with ICH requirements. Atorvastatin has a limit of quantification of 0.75 ng/mL and a limit of detection of 0.25 ng/Ml.^[52] Richard Myles Turner et al. developed and validated a novel HPLC-MS/MS method for quantifying atorvastatin, bisoprolol, and clopidogrel in human plasma, particularly for large cardiovascular patient cohorts. A 2.7 µm Halo C18 column (50 x 2.1 mm ID, 90Å) with gradient elution was used for the chromatographic separation. Water and 0.1% formic acid made up mobile phase A, while acetonitrile and 0.1% formic acid made up mobile phase B. The entire run time was 6.00 minutes, with a flow rate of 500 µL/min. The retention time for atorvastatin was 3.57 minutes. Multiple reaction monitoring (MRM, MS/MS) was used for detection and quantification. The technique proved suitable for extensive clinical research because to its excellent sensitivity, accuracy, and reproducibility.^[53] Venkanna Bayya et al. developed and validated a rapid, selective, and rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of atorvastatin and its active metabolites, o-hydroxy atorvastatin and p-hydroxy atorvastatin, in stabilized plasma. Using a mobile phase of acetonitrile and 0.20% formic acid (65:35 v/v) at a flow rate of 0.60 mL/min and a column temperature was 40 ± 5°C, the chromatographic separation was accomplished by isocratic elution on a Luna C18 column (100 mm x 4.60 mm, 5 μm). To improve sensitivity and selectivity, negative mode electrospray ionization was used. Atorvastatin, p-hydroxy atorvastatin, and o-hydroxy atorvastatin had retention durations of 3.73, 2.34, and 3.33 minutes, respectively. The method demonstrated excellent linearity, precision, accuracy, and stability under various conditions, with high sensitivity and selectivity due to the isocratic elution and negative mode ionization.^[54] Bullen, W.W. et al. developed and validated a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of atorvastatin, ortho-hydroxy atorvastatin, and para-hydroxy atorvastatin in human, dog, and rat plasma. Using YMC J'Sphere H80 C18 column and a mobile phase consisting of acetonitrile and 0.1% acetic acid (70:30, v/v) at a flow rate of 0.2 mL/min, chromatographic separation was carried out. MS/MS using electrospray ionization in the positive ion mode was used to detect the analytes. Retention times for atorvastatin, para-hydroxy atorvastatin, ortho-hydroxy atorvastatin, [d5]-ortho-hydroxy atorvastatin, and [d5]-atorvastatin were 2.27 ± 0.21, 3.36 ± 0.23, 3.54 ± 0.46, 4.12 ± 0.61, and 4.65 ± 0.65 minutes, respectively. With no interfering peaks found during validation, the approach was shown to be appropriate for routine quantification of the substances throughout a concentration range of 0.250 to 25.0 ng/mL.^[55]