**4. Capillary electrophoresis**

Capillary electrophoresis (CE) is an alternative separation technique which is designed to separate species based on their size to charge ratio in an electric field in the interior of a small capillary filled with background electrolyte. Driving forces in CE are electrophoretic migration and the electro-osmotic flow (EOF). CE has become a useful tool in pharmaceutical analysis because of its advantages over other separation techniques, such as high resolution, high selectivity, simplicity, short analysis time, cost efficiency and low consumption of solvents and reagents [102]. Mainly employed CE modes for drug analysis are capillary zone electrophoresis (CZE) based on charge-to-mass ratio and micellar electrokinetic chromatography (MEKC) based on chromatographic partition of analytes between micelles and background electrolyte. MEKC is the most appropriate electrophoretic technique for impurity profiling because the neutral compounds and charged components that have similar electrophoretic mobilities can be separated simultaneously [103]. CE is currently recommended in several pharmacopoeias. Principal advantage of CE over wellestablished and widely used HPLC technique is its ability to deliver high efficiency in short analysis times [104]. However, CE methods proposed for the determination of statin drugs are scarce.

412 Chromatography – The Most Versatile Method of Chemical Analysis

and losartan, atenolol, acetylsalicylic acid [99].

**4. Capillary electrophoresis** 

drugs.

Furthermore, a LC/MS/MS method for separation of fluvastatin from its *threo* isomers

The advantage of the methods for simultaneous determination of several co-administered drugs is that the one sample preparation and one chromatographic run are required for monitoring therapeutic levels of several drugs. Therefore, these methods could be useful in daily routine sample handling, when many samples from patients taking different drugs together with HMG-CoA reductase inhibitors are analyzed in clinical laboratories. Recently, several chromatographic methods have been developed for the quantification of statin drugs in combination with other drugs, most of them are commonly used in treatment of cardiovascular disease: atenolol, spironolactone, glibenclamide [61], metoprolol succinate [72], valsartan and chlorthalidone [76, 87], timolol maleate, diclofenac sodium [81], fenofibric acid [85], ezetimibe [91], ceftriaxone [92], acetylsalicylic acid [94], amlodipine [98]

Recently, several papers were published regarding prediction of statins' pharmacokinetics. In our work the usefulness of reversed-phase high performance chromatography in building models that would allow the prediction of pharmacokinetics parameters of statins was evaluated [100]. In order to get better insight into the nature of their chromatographic behavior, the retention times were measured using octyl and octadecyl chromatographic columns. Obtained chromatographic data were compared with pharmacokinetic parameters predicted by use of 17 different computer programs. Significant correlations were found between chromatographic data and lipophilicity of statins. In addition, with the combine set of descriptors (chromatographic data, solubility, quantum chemical and topological indices) the highly significant correlations with pharmacokinetic parameters have been found, which confirms the utility of HPLC technique for prediction of pharmacokinetic behavior of statin

In order to predict the bioavailability of statins, the association mechanism with phosphatidylcholine using immobilized artificial membrane high performance liquid chromatography technique was studied. Moreover, the thermodinamic driving forces for the statin molecules with phosphatidylcholine monolayers were analyzed in detail [101].

Capillary electrophoresis (CE) is an alternative separation technique which is designed to separate species based on their size to charge ratio in an electric field in the interior of a small capillary filled with background electrolyte. Driving forces in CE are electrophoretic migration and the electro-osmotic flow (EOF). CE has become a useful tool in pharmaceutical analysis because of its advantages over other separation techniques, such as high resolution, high selectivity, simplicity, short analysis time, cost efficiency and low consumption of solvents and reagents [102]. Mainly employed CE modes for drug analysis are capillary zone electrophoresis (CZE) based on charge-to-mass ratio and micellar electrokinetic chromatography (MEKC) based on chromatographic partition of analytes between micelles and background electrolyte. MEKC is the most appropriate electrophoretic

metabolites to support a bioequivalence study has been developed [97].

CE has been applied for determination of pravastatin in fermentation broth in order to optimize its production in bioreactors [105]. Pravastatin is produced in two-step fermentation. In the first step, mevastatin is produced by *P. citrinum*, and in the second step, bioconverted to pravastatin by *S. carbophylus*. The method successfully separated pravastatin from interfering matrix, mevastatin and 6-*epi* pravastatin. Its determination in production media was also performed using two HPLC methods. All three proposed methods had runtimes under 1 min. However two HPLC methods, performed on a particle and a monolithic LC column had superior sensitivity compared to MEKC, with LOD around 0.01 ng/mL, 0.2 ng/mL and 20 ng/mL, respectively.

We have developed CZE method for determination of pravastatin in pharmaceutical dosage form [106]. Rapid migration of negatively charged pravastatin molecule was obtained in alkaline buffer by the application of electric field of 30 kV. The alkaline buffer generated strong EOF that enabled determination of a fully charged drug molecule within 2.5 min. Pravastatin retention time is about 21 min in the assay procedure listed in European Pharmacopoeia (Ph. Eur.) using the HPLC with UV detection. Relatively short analysis time is the main advantage of the CZE method developed. Pravastatin is administered to patients in its active form as the hydroxy acid sodium salt. However, the drug exists in solution with its lactone equilibrium product reversibly formed at acidic pH. Pravastatin is also susceptible to an isomerization reaction which is relatively rapid [107]. The MEKC method was established to separate the drug and its degradation products in acidic media. Introduction of sodium dodecyl sulphate (SDS) in the background electrolyte solution plays a key role in the separation of negatively charged and neutral species. The proposed method allows baseline separation of pravastatin, C-6 epimer of pravastatin and their corresponding lactone forms that appear as interconversion products depending on the pH value. The migration times of degradation compounds ranged from 2.8 to 6.2 min. The above mentioned interconversion compounds of pravastatin represent its related impurities defined in Ph. Eur. and are also potential biotransformation products. CE has also been applied to the screening of anionic impurities in bulk drug [108].

The application of CE to rapidly quantitate lovastatin production levels by *Aspergillus terreus* mutants has been described [109]. The fermentation broths of thousands of mutated strains were efficiently and inexpensively screened for increased lovastatin production by the developed high-throughput method. Determination of lovastatin in the presence of its oxidation products after exposure to an oxidative atmosphere has been carried out using CE technique [110]. The method developed is suitable for the routine analysis of lovastatin.

The quantitative analysis of lovastatin in urine samples based on CE has significance for the control of clinical therapy [111]. The concentration sensitivity is poor in CE because of the short optical path length limited by the inner diameter of the capillary and small volume of sample injected. Such low sensitivity has hampered the use of this method in clinical drug monitoring. However, the sensitivity was enhanced by using a simple stacking method for the determination of trace lovastatin in biologic fluids.

A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 415

of atorvastatin and fenofibrate has better sensitivity and runtime of 3 min but the linearity,

CE method was also developed for the separation and simultaneous determination of atorvastatin and amlodipine in their combination formulations [116]. Degradation products produced as a result of stress studies did not interfere with the detection of both drugs and

The CE method was developed for the enantiomeric purity determination of fluvastatin enantiomers [117]. Its principle involves the formation of diastereoisomer complexes after addition of neutral cyclodextrin to the running buffer. Fluvastatin enantiomers were separated on an uncoated fused silica with 100 mM borate solution containing 30 mg/mL of (2-hydroxypropyl)-β-cyclodextrin as running buffer and fenoprofen as an internal standard. The limit of detection and quantification for (+)-3R, 5S and (-)-3S, 5R-fluvastatin were 1.5 μg/mL and 2.5 μg/mL, respectively. Compared to chiral LC separations, CE analyses are cheaper (no chiral column, no solvent, low consumption of chiral selector) and peak

There is only one CE method for quantification of rosuvastatin [118]. Currently, for rosuvastatin only a limited number of analytical methods are reported in literature. This is due to the fact that rosuvastatin is a new statin introduced in the EU in 2002 and approved

Using neutral β-cyclodextrin as chiral selector, the CZE method has been established for the chiral separation of pitavastatin calcium enantiomers [119]. Pitavastatin is a novel statin that potentially represents an important addition to the cardiovasculary therapy. In view of this, simple and efficient capillary electrophoretic methods for the determination of rosuvastatin

Since statins differ in their structure, analytical methods for their determination are developed individually. In fact, since statin drugs are never co-administered together during treatment of hyperlipimidemia, some authors even argued that there is no need for their simultaneous analysis. However, lately papers have been published that propose analytical methods that enable separation, identification and quantitative determination for two and even all six statins simultaneously in a single run. This kind of method would allow determination of any statin available on the market without the need of developing a new, separate, individual method for each statin, and could by used for simultaneous analysis of

A HPTLC method was published using precoated silica gel 60F 254 aluminum sheets and detection carried out at 239, 238 and 310 nm for determination of simvastatin, pravastatin and rosuvastatin in tablet dosage forms, respectively [120]. Far to our knowledge first HPLC-PDA method for simultaneous analysis of atorvastatin, lovastatin, pravastatin,

LOQ and LOD was established only for atorvastatin lactone [43].

the assay can thus be considered stability indicating.

efficiencies are higher by one order of magnitude.

**5. Simultaneous analysis of statin drugs** 

pharmaceutical dosage forms or in routine clinical monitoring.

and pitavastatin are highly required.

in the US in 2003.

The CZE method was developed for the separation and determination of lovastatin as active ingredient in the red yeast rice product [112]. Prior to determination, lovastatin was extracted from capsule by ethanol. In this study, high pH (10.5) was selected in order to convert lovastatin to its acidic form completely. However, earlier reported studies revealed that lovastatin and lovastatin hydroxy acid are the two main components which contribute to up to 90% of the total quantity of monacolins in the read yeast rice [13]. Hence, the main disadvantage of the proposed CE method is that the content of the main components contributing to the pharmacology effect in red yeast rice supplement was not determined individually.

Only one CE method for the analysis of simvastatin is available till date [113]. This method was developed for the quanitication of both lovastatin and simvastatin in pharmaceutical dosage forms.

In the literature, CZE method has been reported for determination of atorvastatin [114]. The separation was optimized on capillary, but it was further miniaturized to a microchip platform with linear imaging UV detection. Even though CE is a rather good alternative for evaluation of impurity profile and enantiomeric purity of a drug, it is not enough applied. Therefore, we have developed a new MEKC method for separation and simultaneous quantitation of atorvastatin and its related substances diastereomer-atorvastatin, desfluoro atorvastatin, atorvastatin methyl ester and atorvastatin lactone [115]. The separation was carried out in an extended light path capillary in order to improve sensitivity at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM SDS and 20% (*v/v*) methanol. Separation of neutral compounds from each other requires partitioning into charged micelles that migrate at a different rate from the EOF. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to atorvastatin as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100-1200 μg/mL for atorvastatin and 1.0-12.5 μg/mL for related substances. The applicability of the proposed MEKC method to the assay of atorvastatin in the presence of its related substances was investigated by analyzing the bulk drug provided by different manufacturers and various commercial formulations. The use of very low volumes of electrolyte (μL) and samples (nL) make the new MEKC procedures very interesting for determination of atorvastatin, purity evaluation and quantification of drug-related substances in a single analysis. The drawback of the proposed MEKC method is lower sensitivity compared to one obtained by RP-LC method for the same related substances [45]. The published UPLC method for simultaneous determination of atorvastatin and fenofibrate has better sensitivity and runtime of 3 min but the linearity, LOQ and LOD was established only for atorvastatin lactone [43].

CE method was also developed for the separation and simultaneous determination of atorvastatin and amlodipine in their combination formulations [116]. Degradation products produced as a result of stress studies did not interfere with the detection of both drugs and the assay can thus be considered stability indicating.

The CE method was developed for the enantiomeric purity determination of fluvastatin enantiomers [117]. Its principle involves the formation of diastereoisomer complexes after addition of neutral cyclodextrin to the running buffer. Fluvastatin enantiomers were separated on an uncoated fused silica with 100 mM borate solution containing 30 mg/mL of (2-hydroxypropyl)-β-cyclodextrin as running buffer and fenoprofen as an internal standard. The limit of detection and quantification for (+)-3R, 5S and (-)-3S, 5R-fluvastatin were 1.5 μg/mL and 2.5 μg/mL, respectively. Compared to chiral LC separations, CE analyses are cheaper (no chiral column, no solvent, low consumption of chiral selector) and peak efficiencies are higher by one order of magnitude.

There is only one CE method for quantification of rosuvastatin [118]. Currently, for rosuvastatin only a limited number of analytical methods are reported in literature. This is due to the fact that rosuvastatin is a new statin introduced in the EU in 2002 and approved in the US in 2003.

Using neutral β-cyclodextrin as chiral selector, the CZE method has been established for the chiral separation of pitavastatin calcium enantiomers [119]. Pitavastatin is a novel statin that potentially represents an important addition to the cardiovasculary therapy. In view of this, simple and efficient capillary electrophoretic methods for the determination of rosuvastatin and pitavastatin are highly required.

## **5. Simultaneous analysis of statin drugs**

414 Chromatography – The Most Versatile Method of Chemical Analysis

the determination of trace lovastatin in biologic fluids.

individually.

dosage forms.

The quantitative analysis of lovastatin in urine samples based on CE has significance for the control of clinical therapy [111]. The concentration sensitivity is poor in CE because of the short optical path length limited by the inner diameter of the capillary and small volume of sample injected. Such low sensitivity has hampered the use of this method in clinical drug monitoring. However, the sensitivity was enhanced by using a simple stacking method for

The CZE method was developed for the separation and determination of lovastatin as active ingredient in the red yeast rice product [112]. Prior to determination, lovastatin was extracted from capsule by ethanol. In this study, high pH (10.5) was selected in order to convert lovastatin to its acidic form completely. However, earlier reported studies revealed that lovastatin and lovastatin hydroxy acid are the two main components which contribute to up to 90% of the total quantity of monacolins in the read yeast rice [13]. Hence, the main disadvantage of the proposed CE method is that the content of the main components contributing to the pharmacology effect in red yeast rice supplement was not determined

Only one CE method for the analysis of simvastatin is available till date [113]. This method was developed for the quanitication of both lovastatin and simvastatin in pharmaceutical

In the literature, CZE method has been reported for determination of atorvastatin [114]. The separation was optimized on capillary, but it was further miniaturized to a microchip platform with linear imaging UV detection. Even though CE is a rather good alternative for evaluation of impurity profile and enantiomeric purity of a drug, it is not enough applied. Therefore, we have developed a new MEKC method for separation and simultaneous quantitation of atorvastatin and its related substances diastereomer-atorvastatin, desfluoro atorvastatin, atorvastatin methyl ester and atorvastatin lactone [115]. The separation was carried out in an extended light path capillary in order to improve sensitivity at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM SDS and 20% (*v/v*) methanol. Separation of neutral compounds from each other requires partitioning into charged micelles that migrate at a different rate from the EOF. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to atorvastatin as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100-1200 μg/mL for atorvastatin and 1.0-12.5 μg/mL for related substances. The applicability of the proposed MEKC method to the assay of atorvastatin in the presence of its related substances was investigated by analyzing the bulk drug provided by different manufacturers and various commercial formulations. The use of very low volumes of electrolyte (μL) and samples (nL) make the new MEKC procedures very interesting for determination of atorvastatin, purity evaluation and quantification of drug-related substances in a single analysis. The drawback of the proposed MEKC method is lower sensitivity compared to one obtained by RP-LC method for the same related substances [45]. The published UPLC method for simultaneous determination

Since statins differ in their structure, analytical methods for their determination are developed individually. In fact, since statin drugs are never co-administered together during treatment of hyperlipimidemia, some authors even argued that there is no need for their simultaneous analysis. However, lately papers have been published that propose analytical methods that enable separation, identification and quantitative determination for two and even all six statins simultaneously in a single run. This kind of method would allow determination of any statin available on the market without the need of developing a new, separate, individual method for each statin, and could by used for simultaneous analysis of pharmaceutical dosage forms or in routine clinical monitoring.

A HPTLC method was published using precoated silica gel 60F 254 aluminum sheets and detection carried out at 239, 238 and 310 nm for determination of simvastatin, pravastatin and rosuvastatin in tablet dosage forms, respectively [120]. Far to our knowledge first HPLC-PDA method for simultaneous analysis of atorvastatin, lovastatin, pravastatin,

rosuvastatin and simvastatin was reported for determination in pharmaceutical formulations and *in vitro* metabolism studies [121]. An uncommon gradient method using 3 mobile phase reservoirs was employed. Downside of the method is relatively long analysis time of 40 minutes and fluvastatin not being included in the simultaneous analysis.

A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 417

We have introduced a universal MEKC method with diode-array detection for the simultaneous and short-time analysis of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin in a single run [127]. Base hydrolysis was used to open lactone ring of lovastatin and simvastatin, administered as lactone prodrugs, in order to transform these compounds to the corresponding acid forms before analysis. This approach offered shorter analysis time due to a decrease of the migration times of negatively charged statin drugs in comparison to neutral lactone forms. The first step in CE method development for optimizing the separation of ionisable statin molecules was the selection of the buffer pH, which determined the extent of ionization and mobility of each drug. As reported in the literature, statins with β-hydroxy acid forms have pKa values between 4.1 and 4.6 [128] and statin molecules are completely in anionic forms above pH 6. Surfactant was added to the electrolyte to improve the selectivity of the separation. With SDS, negatively charged statins molecules were not strongly attracted to the micelles, and drug molecules were separated as a result of differences in their electrophoretic mobilities and lipophilicity. The addition of an organic modifier in the presence of SDS in the electrolyte solution played a key role in the separation of statin molecules. The addition of an organic modifier changes the selectivity and migration times due to the change in electrolyte viscosity, dielectric constant and the zeta potential. The SDS micelles and methanol in the concentration of 10% *v/v* added to the borate buffer (pH 9.5) were employed in order to reduce the analysis time while maintaining good resolution between all six statins. The new developed MEKC method enabled a powerful separation and simultaneous, simple and rapid determination of six statins in 5 min (Figure 3). The method developed was successfully applied to analysis of six

different pharmaceutical dosage forms of statin drugs.

**Figure 3.** Electropherogram of a simultaneous analysis of statins

**rosuvastatin**

**IS**

**pravastatin**

**0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5**

**atorvastatin**

**fluvastatin**

**lovastatin**

**simvastatin**

**Time (min)**

**-5**

**0**

**5**

**10**

**Absorbance (mAU)**

**15**

**20**

**25**

An interesting method for pharmaceutical analysis of atorvastatin, simvastatin and lovastatin using a charged aerosol detector (CAD) was published [122]. CAD is a universal detector for HPLC that operates regardless of the physiochemical and spectral properties of non-volatile analytes. It can provide data complementary to UV or MS detectors. The eluent from the HPLC column is first nebulized and then charged. A highly sensitive electrometer generates a signal proportional to the analyte quantity. Although CAD is considered as a non-linear detector, the authors found a perfectly linear response (R > 0.9995). Sensitivity of the CAD detector was two folds greater then the UV detector; LOD of atorvastatin measured with UV and CAD detectors was 0.17 μg/mL and 0.08 μg/mL, respectively.

A HPLC-UV method for quantification of rosuvastatin, atorvastatin, fluvastatin, lovastatin and simvastatin and four fibrates in pharmaceutical dosage forms was developed [123]. In this paper a simple GC-FID method was also proposed for identification of atorvastatin, lovastatin and simvastatin along four fibrates.

Two stability-indicating HPLC methods for quantitative determination of pravastatin, fluvastatin, atorvastatin and rosuvastatin in pharmaceuticals were developed [124].

Methods for their simultaneous determination in biological samples could provide easy quantification of drug level in human plasma without changes in the chromatographic procedures for individual statin. Despite the fact that these drugs seem to be structurally similar, development of the method for their simultaneous determination in complex biological samples is quite challenging task as they differ significantly in terms of solubility, polarity, stability as well as optic characteristics. Until now several analytical methods have been developed for the determination of statins in biological samples simultaneously, simvastatin and atorvastatin [83], rosuvastatin and atorvastatin [90], simvastatin, pravastatin, rosuvastatin and atorvastatin [92].

Investigation of statins in the environment has become an important issue in the last years due to their large worldwide consumption and their potential adverse effects on animal and human health. Three different preconcentration techniques including solid phase extraction, dispersive liquid–liquid microextraction and stir-bar sorptive extraction have been optimized and compared for the simultaneous analysis of statin drugs in wastewater and river water samples by HPLC coupled to quadrupole-time-of-flight mass spectrometry [125].

Due to low sensitivity of CE, three on-line preconcentration strategies were investigated for the analysis of charged and neutral statins by MEKC [126]. A background electrolyte consisting of 20 mM ammonium bicarbonate buffer (pH 8.50) and 50 mM SDS was used for the separation of all statin molecules including mevastatin. The methods were applied for the analysis of statin analytes in wastewater samples. The more frequently prescribed statins are of environmental concern. Consequently, sensitive methods for investigation of distribution of statin drugs in the environment are very valuable.

We have introduced a universal MEKC method with diode-array detection for the simultaneous and short-time analysis of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin in a single run [127]. Base hydrolysis was used to open lactone ring of lovastatin and simvastatin, administered as lactone prodrugs, in order to transform these compounds to the corresponding acid forms before analysis. This approach offered shorter analysis time due to a decrease of the migration times of negatively charged statin drugs in comparison to neutral lactone forms. The first step in CE method development for optimizing the separation of ionisable statin molecules was the selection of the buffer pH, which determined the extent of ionization and mobility of each drug. As reported in the literature, statins with β-hydroxy acid forms have pKa values between 4.1 and 4.6 [128] and statin molecules are completely in anionic forms above pH 6. Surfactant was added to the electrolyte to improve the selectivity of the separation. With SDS, negatively charged statins molecules were not strongly attracted to the micelles, and drug molecules were separated as a result of differences in their electrophoretic mobilities and lipophilicity. The addition of an organic modifier in the presence of SDS in the electrolyte solution played a key role in the separation of statin molecules. The addition of an organic modifier changes the selectivity and migration times due to the change in electrolyte viscosity, dielectric constant and the zeta potential. The SDS micelles and methanol in the concentration of 10% *v/v* added to the borate buffer (pH 9.5) were employed in order to reduce the analysis time while maintaining good resolution between all six statins. The new developed MEKC method enabled a powerful separation and simultaneous, simple and rapid determination of six statins in 5 min (Figure 3). The method developed was successfully applied to analysis of six different pharmaceutical dosage forms of statin drugs.

416 Chromatography – The Most Versatile Method of Chemical Analysis

lovastatin and simvastatin along four fibrates.

pravastatin, rosuvastatin and atorvastatin [92].

rosuvastatin and simvastatin was reported for determination in pharmaceutical formulations and *in vitro* metabolism studies [121]. An uncommon gradient method using 3 mobile phase reservoirs was employed. Downside of the method is relatively long analysis

An interesting method for pharmaceutical analysis of atorvastatin, simvastatin and lovastatin using a charged aerosol detector (CAD) was published [122]. CAD is a universal detector for HPLC that operates regardless of the physiochemical and spectral properties of non-volatile analytes. It can provide data complementary to UV or MS detectors. The eluent from the HPLC column is first nebulized and then charged. A highly sensitive electrometer generates a signal proportional to the analyte quantity. Although CAD is considered as a non-linear detector, the authors found a perfectly linear response (R > 0.9995). Sensitivity of the CAD detector was two folds greater then the UV detector; LOD of atorvastatin measured with UV and CAD detectors was 0.17 μg/mL and 0.08 μg/mL, respectively.

A HPLC-UV method for quantification of rosuvastatin, atorvastatin, fluvastatin, lovastatin and simvastatin and four fibrates in pharmaceutical dosage forms was developed [123]. In this paper a simple GC-FID method was also proposed for identification of atorvastatin,

Two stability-indicating HPLC methods for quantitative determination of pravastatin,

Methods for their simultaneous determination in biological samples could provide easy quantification of drug level in human plasma without changes in the chromatographic procedures for individual statin. Despite the fact that these drugs seem to be structurally similar, development of the method for their simultaneous determination in complex biological samples is quite challenging task as they differ significantly in terms of solubility, polarity, stability as well as optic characteristics. Until now several analytical methods have been developed for the determination of statins in biological samples simultaneously, simvastatin and atorvastatin [83], rosuvastatin and atorvastatin [90], simvastatin,

Investigation of statins in the environment has become an important issue in the last years due to their large worldwide consumption and their potential adverse effects on animal and human health. Three different preconcentration techniques including solid phase extraction, dispersive liquid–liquid microextraction and stir-bar sorptive extraction have been optimized and compared for the simultaneous analysis of statin drugs in wastewater and river water

Due to low sensitivity of CE, three on-line preconcentration strategies were investigated for the analysis of charged and neutral statins by MEKC [126]. A background electrolyte consisting of 20 mM ammonium bicarbonate buffer (pH 8.50) and 50 mM SDS was used for the separation of all statin molecules including mevastatin. The methods were applied for the analysis of statin analytes in wastewater samples. The more frequently prescribed statins are of environmental concern. Consequently, sensitive methods for investigation of

samples by HPLC coupled to quadrupole-time-of-flight mass spectrometry [125].

distribution of statin drugs in the environment are very valuable.

fluvastatin, atorvastatin and rosuvastatin in pharmaceuticals were developed [124].

time of 40 minutes and fluvastatin not being included in the simultaneous analysis.

**Figure 3.** Electropherogram of a simultaneous analysis of statins
