**3. Bioanalytical methods**

398 Chromatography – The Most Versatile Method of Chemical Analysis

**technique and detector** 

HPLC PDA 237 nm

HPLC UV 240 nm

HPLC DAD 240 nm, ESI-ion trap

UPLC Q-TOF MS

HPLC APCI-CAD MS

HPLC UV 238 nm

HPLC UV 237 nm

UPLC UV 247 nm

UPLC UV 247 nm

HPLC UV 248 nm

HPLC HPTLC

TLC UV 258 nm

HPLC UV 225 nm ESI-QTOF MS **Stationary phase Mobile phase Ref.** 

Gradient elution A: ACN B: 0.1% TFA

acetate (65:35)

gradient elution

gradient elution A: 30%methanol+10 mM ammonium acetate B: 100% methanol + 10 mM ammonium acetate

buffer pH 7

4.5 (55:45)

acetate pH 4.7

(90:10)

pH 3.5

(38:62)

gradient elution

6

ACN:50 mM ammonium

ACN:water (85:15) 26

ACN:ammonium acetate pH

methanol:0.02 M phosphate

ACN:0.1 M phosphate buffer 40

ACN:0.025 M sodium dihydrogen phosphate pH

ACN:0.01 M ammonium

A: Tetrahydrofuran:ACN

B: 0.025M phosphate buffer

0.1% phosphoric acid:ACN

methanol-benzene-glacial acid (19.6:80.0:0.4)

B: phosphate buffer pH 2.3

toluene-methanoltriethylamine (7:3:0.2)

gradient elution A: ACN

15

25

28

32

33

39

43

45

47

48

56

Symmetry C18 (150 x 3.9 mm, 5 μm)

Symmetry Shield RP 18 (250 x 4.6 mm, 5

Acquity BEH C18 (100 x 2.1 mm, 1.7 μm)

Betasil C18 (250 x 4.6

Alltima C18 (150 x 4.6

Perfectsil Target ODS-3 (250 x 4.6 mm, 5 μm)

BEH C18 (50 x 2.1 mm,

BEH C18 (50 x 2.1 mm,

Zorbax XDB C18 Rapid Resolution HT (50 x 4.6 mm, 1.8 μm)

Phenomenex Luna C18 (250 x 4.6 mm, 5

Silica gel 60F254

gel 60 F-254

mm, 5 μm)

Aluminum foil silica

Supelco C8 (250 x 4.6

mm, 5 μm)

1.7 μm)

1.7 μm)

μm)

Chromolith Performance monolitich column RP-18e (250 x 4.6 mm)

μm)

mm)

**Analyt Application Separation** 

indicating study

dosage form

profiling

dosage form

profiling

stability indicating study

stability indicating study

stability indicating study

compounds

combined dosage form

combined dosage form

pharmaceutical dosage form

indicating study

LOV stability

SIM, EZE combined

SIM impurity

SIM combined

PRA impurity

PRA stability

ATO related

ATO, AML

ATO, ASA

ATO, FEN

ATO, RAM

ATO, FEN

ATO, SIM, PRA

There have been three reviews on analytical methods for the determination of HMG-CoA reductase inhibitors in biological samples. The first one, published by Ertürk and coworkers in 2003 [70], reviews bio-analytical methods for lovastatin, simvastatin, pravastatin, fluvastatin and atorvastatin. The second one, published in 2007, is focused only on chromatography-mass spectrometry methods for the quantification of statins in biological samples [10]. In 2008 Nováková and co-workers [17] have published a review on HPLC methods for the determination of simvastatin and atorvastatin in various fields of application, including bioanalytical assays. Since these reviews have been published, a number of bioanalytical methods have been developed for all HMG-CoA reductase inhibitors. Most of the methods published since 2007 were applied for investigation of HMG-CoA reductase inhibitors in human plasma or serum. Far to our knowledge since 2007 only two LC/MS/MS methods for determination of statins in human urine have been developed. The sample preparation procedures and analytical assays for quantification of statins in biological samples are listed in Tables 2 and 3.

## **3.1. Sample preparation**

Sample preparation is a quite tedious but still unavoidable procedure in bioanalitical methods. The objective of this delicate and challenging step is to transfer analyte of interest into a form that is purified, concentrated and compatible with the analytical system. The extraction and enrichment of analytes from the sample matrix are often realized by procedures such as, protein precipitation, liquid-liquid extraction (LLE) and solid-phase extraction (SPE). These conventional sample preparation procedures are still dominating in the preparation of biological samples for determination of statin drugs as well as their metabolites.

A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 401

and aqueous humor from the bovine eyes was proposed [81]. The mobile phase, consisted of acetonitrile and 0.2% triethylamine, was used as extraction solvent. The quite high extraction efficacy of all investigated compounds was obtained using this uncommon

Although LLE is generally considered to be providing cleaner extracts and lower matrix effect than the SPE, lower recovery due to the transfer of a fraction of the organic extract after the extraction may be the main disadvantage of the LLE technique. Moreover, when low concentrations have to be detected it is necessary to use a large solvent volumes and sample preparation becomes time consuming and labor invasive. In order to reduce organic solvent consumption, sample volume and sample preparation time, Apostolou and coworkers [75] have presented a fully automated high-throughput two-step LLE-LC/MS/MS method for the quantification of simvastatin and its acid form using a robotic liquid handling workstation with 96-deepwell plates. Another fully automated high-throughput salting-out (SA) assisted LLE-LC/MS/MS method was introduced by Zhang and co-workers [82]. Due to the compatibility between SALLE and LC/MS/MS, the extracts of simvastatin and its acid from human plasma were injected directly into LC system immediately after sample extraction. In this way extract solvent evaporation was eliminated and consequently sample preparation procedure was simplified. Also, the exposure of the extracts to the room temperature was minimized and hence minimal interconversion between simvastatin and

Among the SPE methods, the reverse phase cartridges have been extensively used for extraction of statins from biological samples. Gonzalez and co-workers [76] have presented a nice work regarding the traditional one-variable-at a time optimization for SPE extraction of fluvastatin together with other drugs from human plasma. The optimization of conditioning and washing solution composition, pH for conditioning and washing step and elution solvent selection were described in details. The SPE procedure has also been used as sample preparation step for quantification of atorvastatin and simvastatin as well as their metabolites in serum from patients with end stage renal disease [83]. In order to obtain the satisfactory and repeatable extraction efficacy and to remove matrix effects, several different reversed-phase SPE sorbents have been tested. The best results were obtained using ZORBAX SPE C-18 (Agilent Technologies) and Discovery DSC-18 SPE (Supelco) cartridges. As ZORBAX SPE C-18 columns were withdrawn from commercial market circulation during optimization of method, further investigations were performed using Discovery DSC-18 SPE cartridges. For the purpose of minimization of the interconversion between lacton and open-ring hydroxy acid forms of simvastatin and atorvastatin, SPE sorbents were conditioned and analytes were eluated with solvents containing 0.1 M acetate ammonium buffer pH 4.5. In the work of Di and co-workers [84) SPE sample preparation procedure was used for determination of pitavastatin with rosuvastatin as internal standard in human plasma. The influence of pH on extraction efficacy of statin drugs was investigated in detail. The authors have pointed out importance of 0.5 M potassium dihydrogenphosphate buffer (pH 4.0) as conditioning reagent for cartridges. At pH lower than 4 both molecules were protonated, leading to a decrease in its partitioning in reversed-phase SPE and recovery. At

extraction solvent.

its acid was achieved.

In most of the methods protein precipitation reagent is used as a dilution solvent for internal standard in order to reduce the number of reagent additions [71-74]. Still, Apostolou and coworkers [75] suggested addition of protein precipitation reagent after the internal standard in order to ensure a more satisfying binding of internal standard molecules with plasma proteins, simulating the binding of proteins with analytes in real human plasma. A number of different protein precipitation reagents were tested [76]. Despite the good recoveries obtained with phosphoric acid, the authors recommended to avoid acidic precipitants due to degradation of fluvastatin in acidic conditions. The highest recoveries were obtained with organic solvents. Although no significant differences were observed between methanol and acetonitrile, the second one was used as it offered a more compact precipitate minimizing the risk of SPE cartridge obstruction.

The simplest way to concentrate the analyte is certainly LLE. Hence, Hamidi and co-workers [73] tested a wide spectrum of organic solvents from various physicochemical categories with different volume fractions as well as combinations for extraction of lovastatin from human plasma. The best extraction efficacy was obtained using diethyl ether as extraction solvent. The same solvent was used for extraction of pitavastatin from human plasma [77]. An addition of hidrocloric acid to the plasma samples afore the extraction procedure was shown to be necessary in order to obtain the non-ionized form of analyte which considerably improved extraction efficacy. Assays employing LLE with ethyl acetate [78] and ethyl ether [79] as extraction solvents for determination of rosuvastatin in plasma samples were already published with extraction recoveries of 74 and 69%, respectively. However, in the preliminary study by Lan and co-workers [80], it was found that the extraction recovery of rosuvastatin from plasma in most common organic solvents, such as above mentioned ethyl acetate and ethyl ether, was less than 20%, resulting in an insufficient, imprecise and inaccurate extraction procedure. The authors presumed that low extraction efficacy of rosuvastatin was due to its extremely low water solubility. However, a carboxyl group in its structure forms a salt with calcium ion which indicates that rosuvastatin was apt to ionization. The application of ion paring with tetrabutyl ammonium hydroxide was suggested for improvement of rosuvastatin solubility and subsequently extraction efficacy. Finally, using ion paring LLE, extraction efficacy of rosuvastatin in ethyl acetate was improved from around 10% to more than 50%. A somewhat unusual LLE method for determination of timolol maleate, rosuvastatin and diclofenac in human plasma and aqueous humor from the bovine eyes was proposed [81]. The mobile phase, consisted of acetonitrile and 0.2% triethylamine, was used as extraction solvent. The quite high extraction efficacy of all investigated compounds was obtained using this uncommon extraction solvent.

400 Chromatography – The Most Versatile Method of Chemical Analysis

Sample preparation is a quite tedious but still unavoidable procedure in bioanalitical methods. The objective of this delicate and challenging step is to transfer analyte of interest into a form that is purified, concentrated and compatible with the analytical system. The extraction and enrichment of analytes from the sample matrix are often realized by procedures such as, protein precipitation, liquid-liquid extraction (LLE) and solid-phase extraction (SPE). These conventional sample preparation procedures are still dominating in the preparation of biological samples for determination of statin drugs as well as their

In most of the methods protein precipitation reagent is used as a dilution solvent for internal standard in order to reduce the number of reagent additions [71-74]. Still, Apostolou and coworkers [75] suggested addition of protein precipitation reagent after the internal standard in order to ensure a more satisfying binding of internal standard molecules with plasma proteins, simulating the binding of proteins with analytes in real human plasma. A number of different protein precipitation reagents were tested [76]. Despite the good recoveries obtained with phosphoric acid, the authors recommended to avoid acidic precipitants due to degradation of fluvastatin in acidic conditions. The highest recoveries were obtained with organic solvents. Although no significant differences were observed between methanol and acetonitrile, the second one was used as it offered a more compact precipitate minimizing

The simplest way to concentrate the analyte is certainly LLE. Hence, Hamidi and co-workers [73] tested a wide spectrum of organic solvents from various physicochemical categories with different volume fractions as well as combinations for extraction of lovastatin from human plasma. The best extraction efficacy was obtained using diethyl ether as extraction solvent. The same solvent was used for extraction of pitavastatin from human plasma [77]. An addition of hidrocloric acid to the plasma samples afore the extraction procedure was shown to be necessary in order to obtain the non-ionized form of analyte which considerably improved extraction efficacy. Assays employing LLE with ethyl acetate [78] and ethyl ether [79] as extraction solvents for determination of rosuvastatin in plasma samples were already published with extraction recoveries of 74 and 69%, respectively. However, in the preliminary study by Lan and co-workers [80], it was found that the extraction recovery of rosuvastatin from plasma in most common organic solvents, such as above mentioned ethyl acetate and ethyl ether, was less than 20%, resulting in an insufficient, imprecise and inaccurate extraction procedure. The authors presumed that low extraction efficacy of rosuvastatin was due to its extremely low water solubility. However, a carboxyl group in its structure forms a salt with calcium ion which indicates that rosuvastatin was apt to ionization. The application of ion paring with tetrabutyl ammonium hydroxide was suggested for improvement of rosuvastatin solubility and subsequently extraction efficacy. Finally, using ion paring LLE, extraction efficacy of rosuvastatin in ethyl acetate was improved from around 10% to more than 50%. A somewhat unusual LLE method for determination of timolol maleate, rosuvastatin and diclofenac in human plasma

**3.1. Sample preparation** 

the risk of SPE cartridge obstruction.

metabolites.

Although LLE is generally considered to be providing cleaner extracts and lower matrix effect than the SPE, lower recovery due to the transfer of a fraction of the organic extract after the extraction may be the main disadvantage of the LLE technique. Moreover, when low concentrations have to be detected it is necessary to use a large solvent volumes and sample preparation becomes time consuming and labor invasive. In order to reduce organic solvent consumption, sample volume and sample preparation time, Apostolou and coworkers [75] have presented a fully automated high-throughput two-step LLE-LC/MS/MS method for the quantification of simvastatin and its acid form using a robotic liquid handling workstation with 96-deepwell plates. Another fully automated high-throughput salting-out (SA) assisted LLE-LC/MS/MS method was introduced by Zhang and co-workers [82]. Due to the compatibility between SALLE and LC/MS/MS, the extracts of simvastatin and its acid from human plasma were injected directly into LC system immediately after sample extraction. In this way extract solvent evaporation was eliminated and consequently sample preparation procedure was simplified. Also, the exposure of the extracts to the room temperature was minimized and hence minimal interconversion between simvastatin and its acid was achieved.

Among the SPE methods, the reverse phase cartridges have been extensively used for extraction of statins from biological samples. Gonzalez and co-workers [76] have presented a nice work regarding the traditional one-variable-at a time optimization for SPE extraction of fluvastatin together with other drugs from human plasma. The optimization of conditioning and washing solution composition, pH for conditioning and washing step and elution solvent selection were described in details. The SPE procedure has also been used as sample preparation step for quantification of atorvastatin and simvastatin as well as their metabolites in serum from patients with end stage renal disease [83]. In order to obtain the satisfactory and repeatable extraction efficacy and to remove matrix effects, several different reversed-phase SPE sorbents have been tested. The best results were obtained using ZORBAX SPE C-18 (Agilent Technologies) and Discovery DSC-18 SPE (Supelco) cartridges. As ZORBAX SPE C-18 columns were withdrawn from commercial market circulation during optimization of method, further investigations were performed using Discovery DSC-18 SPE cartridges. For the purpose of minimization of the interconversion between lacton and open-ring hydroxy acid forms of simvastatin and atorvastatin, SPE sorbents were conditioned and analytes were eluated with solvents containing 0.1 M acetate ammonium buffer pH 4.5. In the work of Di and co-workers [84) SPE sample preparation procedure was used for determination of pitavastatin with rosuvastatin as internal standard in human plasma. The influence of pH on extraction efficacy of statin drugs was investigated in detail. The authors have pointed out importance of 0.5 M potassium dihydrogenphosphate buffer (pH 4.0) as conditioning reagent for cartridges. At pH lower than 4 both molecules were protonated, leading to a decrease in its partitioning in reversed-phase SPE and recovery. At

pH higher than 4, the carboxylic group in both pitavastatin and rosuvastatin undergo ionization, which also resulted in a decrease in the recovery for the same reason. Furthermore, it was found that pitavastatin degradation was much faster at lower than at high pHs. Also, it was found that pitavastatin was sensitive to sunlight. It was recommended to minimize the exposure of samples to sunlight as well as to dissolve the dried extract rather in methanol and water than in mobile phase containing formic acid.

A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 403

recently developed columns based on BEH particles technology were employed in several methods [83, 86, 87]. Only in one assay reversed-phase C8 chromatographic column was used [88]. Unusually, reversed-phase narrow bore phenyl column was employed for investigation of atorvastatin, rosuvastatin and their metabolites [74, 89]. The length and diameter of columns differed fairly from 50 to 250 mm and from 2.0 to 4.6 mm, respectively. Although in most of the cases columns with particle size 5 μm were used, several authors preferred columns with smaller particles in order to obtain better peak shapes, resolution and thus shorter analysis time [72, 82, 83, 86, 87]. Analytical run times have been very

The selection of mobile phase was quite a challenging task in all investigations. In most of the methods acetonitrile or methanol were present in the mobile phase as organic solvent. The percentage of organic solvents was optimized such that the retention times of analytes were kept as short as possible. In most assays percentage of organic solvent was quite high, usually more than 70%. The majority of publications emphasize the pH as the most critical variable for separation of the statin drugs [76, 82, 84]. In order to minimize the

The influence of mobile phase pH on retention of atorvastatin and rosuvastatin has been investigated [90]. Since both of the analytes are acidic compounds, their retention on the reversed-phase column was expected to be pH dependant. When pH of the mobile phase was decreased from 4.0 to 3.0, the retention times of the analytes decreased unexpectedly and with further decreases in the pH to 2.0 the retention times increased once again. This behavior was explained by a change in binding of the analytes to the stationary phase and also changes in the solubility of the analytes in the mobile phase. The pH 3.0 was chosen as optimum pH because of the reasonable retention times while the resolution between peaks,

The pH of mobile phase was also a critical variable for the separation of the fluvastatin from valsartan and its metabolite during the optimization of LC/PDA/FLD method [76]. The pH of the mobile phase was limited by the native fluorescence of valsartan and its metabolite, which disappears in the basic form (p*K*a = 3.7). On the other hand, spectrophotometric studies showed that fluvastatin degradation was accelerated in acidic conditions. Mobile phases with different formic acid/formate proportions were tested in order to establish the range where fluvastatin was stable and valsartan and its metabolites kept their fluorescence. 0.01% formic acid/10 mM ammonium formate (pH 4.1) was finally chosen as appropriate buffer. Uncommon pH was used for quantification of lovastatin in human plasma [73]. Mobile phase consisted of acetonitrile and 0.05 M phosphate buffer with pH 7, adjusted with

The flow rate of the mobile phase was in range from 0.2 up to 1.5 mL/min. In all of the assays the flow rate did not change during the chromatographic analysis except in the

The chromatographic separation of most of the methods was performed at room temperature. In order to shorten analysis time, in the several cases the column temperature

reference [76] where the flow rate was gradually changed after three minutes.

interconversion, it is critical to maintain pH of mobile phase between 4 and 5.

variable, the shortest 2 min, the longest about 20 min.

as well as peak shapes, were satisfactory.

phosphoric acid.

To reduce the time of sample preparation, Mertens and co-workers [85] have used an automated SPE on disposable extraction cartridges to isolate pravastatin and its metabolites together with fenofibric acid, another lipid-regulating agent, from the human plasma and to prepare cleaner samples before injection and analysis in the LC/DAD/MS/MS system. Different kinds of disposable extraction cartridges containing bonded silicas of different polarities (ethyl, endcapped ethyl, octyl, endcapped octyl, octadecyl, endcapped octadecyl and cyanopropyl) were tested. The best recoveries for all investigated compounds were reported when disposable extraction cartridges filled with octyl functionalized silica sorbent were used.

Unfortunately, conventional SPE and LLE approaches are multi-step, time-consuming and the sample required for analyses as well as the consumption of organic solvent are quite high, particularly in case of LLE. A solvent-minimized sample preparation approach has been popular in last decades, therefore Farahani and co-workers [71] have published liquidliquid-liquid microextraction procedure (LLLME), a miniaturized format of LLE, for determination of atorvastatin in human plasma. A number of factors affecting the microextraction efficiency were studied in detailed and the optimized conditions were established. They have obtained quite high extraction efficacy of atorvastatin from human plasma using proposed sample preparation procedure. Vlčková and co-workers [86] have developed fast and simple extraction procedure using microextraction by packed sorbent (MEPS) for sample purification and concentration of atorvastatin and its metabolites from human serum. Briefly, MEPS is a miniaturization of conventional SPE, but it differs from commercial SPE by fact that packing is inserted directly into the syringe, not into a separate column. In addition, they have compared a previously described [83] SPE procedure for extraction of atorvastatin and its metabolites from human serum with newly developed MEPS approach. The results of samples treated by SPE and MEPS were compared by means of Student *t*-test. The difference between obtained concentrations was statistically not significant. Hence, MEPS procedure was found to be simpler and faster sample preparation technique using smaller volume of sample, which is regardful to the patients and smaller volume of solvents, which is environmentally friendly.
