**3.2. Liquid chromatography**

## *3.2.1. High performance liquid chromatography*

The high performance liquid chromatography has become the method of choice for bioanalytical methods. Generally, in the HPLC methods reversed-phase C18 chromatographic columns were used for analysis of statin drugs in biological fluids. The 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 variable, the shortest 2 min, the longest about 20 min.

402 Chromatography – The Most Versatile Method of Chemical Analysis

volume of solvents, which is environmentally friendly.

*3.2.1. High performance liquid chromatography* 

**3.2. Liquid chromatography** 

were used.

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.

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

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

The high performance liquid chromatography has become the method of choice for bioanalytical methods. Generally, in the HPLC methods reversed-phase C18 chromatographic columns were used for analysis of statin drugs in biological fluids. The 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 interconversion, it is critical to maintain pH of mobile phase between 4 and 5.

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, as well as peak shapes, were satisfactory.

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 phosphoric acid.

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

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


A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 405

was maintained above 30 °C [76, 77, 81, 83, 86, 87]. The effect of column oven temperatures on the analysis of atorvastatin and rosuvastatin in the range 25 to 35 °C was investigated and best results were observed at 25 °C in terms of retention factor and resolution [90]. Increasing temperature above 25 °C resulted in the rapid elution of rosuvastatin close to the

LC/DAD methods are rarely sensitive enough for quantification of statins as well as their metabolites in human plasma samples due to the poor UV-absorption properties of statin molecules. Furthermore, the levels of statins and their metabolites in biological fluids are very low due to low amount of drug reaching the systemic circulation. Their typical plasma concentrations are in ng/mL levels. However, several sensitive LC/DAD methods for determination of pravastatin [31], atorvastatin [71], lovastatin [73], rosuvastatin [81], and atorvastatin with rosuvastatin [90] have been developed with limit of quantification (LOQ) in range of 1 - 10 ng/mL. Less sensitive LC/DAD method for quantification of lovastatin in human plasma was developed [91]. The LOQ value for lovastatin was relatively high, 400 ng/mL. Another even less sensitive LC/DAD method for quantification of several HMG-CoA reductase inhibitors in human plasma was developed by Sultana and co-workers [92]. The LOQ values were between 376 and 1006 ng/mL. In fact, both of these methods were not

Fluorescence detection has not been widely employed in the determination of HMG-CoA reductase inhibitors, as most of statins do not possess a natural native fluorescence. Still, Gonzalez and co-workers [76] have developed a SPE-HPLC/PDA/FLD method for determination of fluvastatin and valsartan in human plasma. Comparing results obtained with spectrophotometic and fluorimetric detector superior selectivity and sensitivity by

Recently UPLC is becoming a leading chromatographic technique in modern bio-analytical methods. Nováková and co-workers [83] have investigated its potential in combination with MS/MS detection for the fast, sensitive, reliable and selective detection of atorvastatin and simvastatin together with their main metabolites and interconversion products in human serum. Iriarte and co-workers [87] have investigated UPLC technique as a faster alternative to HPLC for simultaneous analysis of fluvastatin and other drugs usually prescribed in cardiovascular therapy. Acquity UPLC Columns Calculator software was used for transfer

The UPLC technology has significantly improved the method optimization process since shorter analysis and re-equilibration times allowed a greater number of experimental testing conditions than with a conventional HPLC. The sample volume required was much lower than in HPLC method. Furthermore, shorter analysis time together with slower flow rates reduced the organic solvent consumption. The sharper and higher chromatographic peaks, thereby improved peak capacity, was obtained using UPLC technology. Still, the sensitivity of UPLC method was found

to be analyte dependent as the improvement was not achieved for all analytes.

solvent front.

used on real plasma samples.

fluorescence detection of fluvastatin could be perceived.

*3.2.2. Ultra performance liquid chromatography* 

of previously developed HPLC method [76].

SIM-simvastatin, PRA-pravastatin, FLU-fluvastatin, ATO-atorvastatin, ROS-rosuvastatin, PIT-pitavastatin, FFAfenofibric acid, TIM-timolol maleate, MET-metoprolol, DIC-diclofenac, VAL-valsartan, ASA-acetylsalycilic acid, CLTchlorthalidone, ACN-acetonitrile, IS-internal standard

**Table 2.** Sample preparation procedures utilized for the determination of statins in biological samples

was maintained above 30 °C [76, 77, 81, 83, 86, 87]. The effect of column oven temperatures on the analysis of atorvastatin and rosuvastatin in the range 25 to 35 °C was investigated and best results were observed at 25 °C in terms of retention factor and resolution [90]. Increasing temperature above 25 °C resulted in the rapid elution of rosuvastatin close to the solvent front.

LC/DAD methods are rarely sensitive enough for quantification of statins as well as their metabolites in human plasma samples due to the poor UV-absorption properties of statin molecules. Furthermore, the levels of statins and their metabolites in biological fluids are very low due to low amount of drug reaching the systemic circulation. Their typical plasma concentrations are in ng/mL levels. However, several sensitive LC/DAD methods for determination of pravastatin [31], atorvastatin [71], lovastatin [73], rosuvastatin [81], and atorvastatin with rosuvastatin [90] have been developed with limit of quantification (LOQ) in range of 1 - 10 ng/mL. Less sensitive LC/DAD method for quantification of lovastatin in human plasma was developed [91]. The LOQ value for lovastatin was relatively high, 400 ng/mL. Another even less sensitive LC/DAD method for quantification of several HMG-CoA reductase inhibitors in human plasma was developed by Sultana and co-workers [92]. The LOQ values were between 376 and 1006 ng/mL. In fact, both of these methods were not used on real plasma samples.

Fluorescence detection has not been widely employed in the determination of HMG-CoA reductase inhibitors, as most of statins do not possess a natural native fluorescence. Still, Gonzalez and co-workers [76] have developed a SPE-HPLC/PDA/FLD method for determination of fluvastatin and valsartan in human plasma. Comparing results obtained with spectrophotometic and fluorimetric detector superior selectivity and sensitivity by fluorescence detection of fluvastatin could be perceived.

## *3.2.2. Ultra performance liquid chromatography*

404 Chromatography – The Most Versatile Method of Chemical Analysis

plasma, bovine aqueous humor

plasma, urine

plasma, urine

chlorthalidone, ACN-acetonitrile, IS-internal standard

**preparation procedure**

ATO plasma PP, LLLME - methanol , HCl,

plasma PP, SPE Phenomenex

serum SPE Supelco

plasma at-SPE Disposable

**Stationary phase** 

plasma PP - methanol:water

plasma PP - 0.1% acetic acid in

Strata-X polymeric C18

plasma LLE - ethyl ether 69–72 79

plasma ion pair LLE - ethyl acetate 47–63 80

PP, LLE - methanol/mobile

plasma SALLE - ACN, 5 M

Discovery DSC-

SupercleanTM LC-18 SPE

18

Tubed

extraction cartridges C8 silica sorbent

serum MEPS C8 ACN:0.1 M

plasma LLE - tertiary butyl

SIM-simvastatin, PRA-pravastatin, FLU-fluvastatin, ATO-atorvastatin, ROS-rosuvastatin, PIT-pitavastatin, FFAfenofibric acid, TIM-timolol maleate, MET-metoprolol, DIC-diclofenac, VAL-valsartan, ASA-acetylsalycilic acid, CLT-

**Table 2.** Sample preparation procedures utilized for the determination of statins in biological samples

LLE - methyl-terc-butyl

SPE Supelco

**PP reagent / LLE reagent / SPE eluent**

/1-octanol

methanol

phase

4.5)

ammonium formate buffer (pH

ACN:0.1 M ammonium acetate buffer pH 4.5 (95:5)

ammonium acetate pH 4.5 (95:5)

ether

methyl ether

methanol plasma

methanol 50–77 85

(1:1)

trichloroacetic acid

ACN/methanol 78–91 76

**Recovery (%)** 

91 71

83-91 72

88–106 74

95–99 81

71–79 82

65-100 83

89–116 86

51–66 94

plasma 70–75 urine 74– 83

84

88

84–88 urine 86– 96

**Ref.** 

**Extracted analytes Matrix Sample** 

SIM, MET IS=propranolol hydrochloride

CLT

ROS

ROS IS=estrone

PIT IS=ROS

ROS + metabolites IS=deuterium labeled

FLU, VAL + metabolite,

IS=candesartan cilexetil

IS=hydrochlorothiazide

SIM, ATO + metabolites IS= deuterium labeled

PRA + metabolites, FFA IS=triamcinolone

ATO + metabolites IS=deuterium labeled

PIT, PIT-lactone IS=racemic *i*-prolact

PRA, ASA IS=furosemide

ROS, TIM, DIC IS=naproxen

SIM, SIM-acid IS=deuterium labeled

> Recently UPLC is becoming a leading chromatographic technique in modern bio-analytical methods. Nováková and co-workers [83] have investigated its potential in combination with MS/MS detection for the fast, sensitive, reliable and selective detection of atorvastatin and simvastatin together with their main metabolites and interconversion products in human serum. Iriarte and co-workers [87] have investigated UPLC technique as a faster alternative to HPLC for simultaneous analysis of fluvastatin and other drugs usually prescribed in cardiovascular therapy. Acquity UPLC Columns Calculator software was used for transfer of previously developed HPLC method [76].

> The UPLC technology has significantly improved the method optimization process since shorter analysis and re-equilibration times allowed a greater number of experimental testing conditions than with a conventional HPLC. The sample volume required was much lower than in HPLC method. Furthermore, shorter analysis time together with slower flow rates reduced the organic solvent consumption. The sharper and higher chromatographic peaks, thereby improved peak capacity, was obtained using UPLC technology. Still, the sensitivity of UPLC method was found to be analyte dependent as the improvement was not achieved for all analytes.

## *3.2.3. Liquid chromatography coupled to tandem mass spectrometry*

In pharmacokinetic investigations of statins LC/MS/MS technique is unequivocally the method of choice. Recently, several procedures were described in the literature taking the advantages of the benefits of mass spectrometry. Both ESI and APCI sources as well as triple quadrupole analyzator were applied in most LC/MS/MS sample analysis.

A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 407

, fewer fragment

operated in the negative detection mode for 1.21 min until simvastatin and lovastatin hydroxy acid forms were eluted from chromatographic column. Afterwards a period of 0.69 min followed in the positive mode during witch simvastatin and lovastatin lacton forms were eluted. Comparing LOQ values for simvastatin acid obtained by these three methods it can be seen that lower LOQ values and thus better sensitivity were obtained in the last two methods. Unfortunately, simvastatin forms various adducts influenced by mobile-phase and matrix composition and such adducts sometimes give higher intensity than protonated molecule [M+H]+, which is an ideal precursor ion for SRM transition and quantification studies. However, Senthamil Selvan and co-workers [72] have observed very high signal of [M+Na]+ in the spectra of simvastatin next to the [M+H]+. Consequently, it was used as precursor ion for quantitation of simvastatin. Also, Zhang and co-workers [82] have used the methylammonium aduct [M+CH3NH4]+ as a parent ion for simvastatin because the

Rosuvastatin has a pyrimidine ring and a carboxylic group in its structure, hence it could be detected either in positive or negative ionization mode. However, the quantification of rosuvastatin in positive ionization mode is more common and was used for determination of rosuvastatin [80] and rosuvastatin together with its metabolites [74], respectively. In the both assays the major ion was protonated molecule [M+H]+ in full-scan mode and principal product ion was at *m/z* 482. Macwan and co-workers [74] have also reported two minor

During the method development, Gao and co-workers [79] also attempted to optimize ESI conditions under positive ionization mode. However, the observed signal intensity was not sensitive enough for determination of expected rosuvastatin's concentrations, especially for low dosage administration. Low sensitivity of positive ionization mode could be explained by a number of fragment ions produced in the product ion spectrum of [M+H]+. In order to improve the sensitivity of the method, the negative ESI detection was taken into consideration. Under negative ESI mode, rosuvastatin produced abundant deprotonated

ions were formed compared with that of [M +H]+. Also, it was pointed out that negative ESI mode produced lower chemical background noise than positive. Comparing LOQ values obtained by these three methods, it can be observed that almost five times lower LOQ value

Pitavastatin has similar structure to rosuvastatin. It contains alkaline nitrogen ion on the quinoline ring and a carboxylic group, therefore positive and negative ionization mode could be also employed. Both of ionization modes for determination of pitavastatin in human plasma and urine by LC/MS/MS method were applied [84]. The results showed that the response intensity of pitavastatin in negative mode was lower and furthermore the response was quite unstable. Pitavastatin was scanned under Q1 MS full-scan mode to determine the parent ion and under Q1/Q3 product ion scan mode to locate the most abundant production. The protonated molecular ion, [M+H]+, was the predominant ion in the Q1 spectrum and was used as the parent ion to obtain the product ion spectra. The most sensitive mass transition was from *m/z* 422.0 to 290.1, which was similar to the MS/MS

molecule [M-H]- at m/z 480. In the product ion mass spectrum of [M-H]-

for rosuvastatin was obtained using negative ESI detection.

adduct ion showed the best signal to noise ratio.

fragments at *m/z* 300 and *m/z* 272.

As it was mentioned above the selection of appropriate mobile phase composition for determination of statins in biological fluids is quite challenging task which is even more complicated when detection and quantification of statins is performed using MS. Only few additives could enable good stability at pH range 4 to 5 as well as volatility and sensitive mass spectrometric response. Therefore, Di and co-workers [84] have pointed out the importance of the formic acid in lowering the pH of mobile phase. In this way pitavastatin was obtained in non-ionized form and a symmetrical peak shape was observed. The concentration of formic acid was optimized not only to maintain a symmetrical peak shape in the chromatographic system but also to render good ionization and fragmentation of pitavastatin in the MS/MS detector. An addition of 0.025% formic acid to the aqueous phase was found to be an important factor for acquiring the high sensitivity of another LC/MS/MS method for determination of pitavastatin in human plasma [77].

Nováková and co-workers [83] have presented a nice example of optimization of the buffer pH and concentration in order to get the best signal to noise ratio of MS detector. Ammonium formate and ammonium acetate at pH 4.0 and 4.5 were tested at the concentration range 0.01 to 10 mM. The best response of atorvastatin and simvastatin was observed at 0.5 mM buffers. The concentrations higher than 5 mM significantly decreased the response of mass spectrometer. On the other hand, the concentrations lower than 0.5 mM were not sufficient to keep buffering capacity and thus had negative influence to the response of mass spectrometer. Ammonium acetate was preferred before ammonium formate because of better peak shapes. Finally, the optimized mobile phase composition was 70% of acetonitrile and 30% of ammonium acetate buffer 0.5 mM (pH 4.0). In most of bioanalytical methods isocratic elution has been utilized, still when more analytes with different polarities were separated, gradient elution had to be applied.

Tandem mass spectrometry detection for identification and quantification of simvastatin and atorvastatin together with their metabolites and lacton/hidroxy acid interconversion forms was employed [83, 86]. All analytes were monitored using electrospray positive ionization (ESI+) mode and for all analytes protonated molecule [M+H]+ was the most intensive ion in mass spectra. Quantification of all analytes was performed using selected reaction monitoring (SRM) and two specific transitions were optimized for each molecule in order to increase selectivity and sensitivity of the method. In the paper published afterwards simvastatin in its lactone form was determined in ESI+ mode, while its hydroxy acid form was determined in ESI mode due to poor sensitivity of hydroxy acid form in positive ion mode [82].

LC/MS/MS method developed by Apostolou and co-workers [75] consisted also of two periods combining both negative and positive ionization modes. The mass spectrometer operated in the negative detection mode for 1.21 min until simvastatin and lovastatin hydroxy acid forms were eluted from chromatographic column. Afterwards a period of 0.69 min followed in the positive mode during witch simvastatin and lovastatin lacton forms were eluted. Comparing LOQ values for simvastatin acid obtained by these three methods it can be seen that lower LOQ values and thus better sensitivity were obtained in the last two methods. Unfortunately, simvastatin forms various adducts influenced by mobile-phase and matrix composition and such adducts sometimes give higher intensity than protonated molecule [M+H]+, which is an ideal precursor ion for SRM transition and quantification studies. However, Senthamil Selvan and co-workers [72] have observed very high signal of [M+Na]+ in the spectra of simvastatin next to the [M+H]+. Consequently, it was used as precursor ion for quantitation of simvastatin. Also, Zhang and co-workers [82] have used the methylammonium aduct [M+CH3NH4]+ as a parent ion for simvastatin because the adduct ion showed the best signal to noise ratio.

406 Chromatography – The Most Versatile Method of Chemical Analysis

*3.2.3. Liquid chromatography coupled to tandem mass spectrometry* 

quadrupole analyzator were applied in most LC/MS/MS sample analysis.

method for determination of pitavastatin in human plasma [77].

different polarities were separated, gradient elution had to be applied.

was determined in ESI-

mode [82].

In pharmacokinetic investigations of statins LC/MS/MS technique is unequivocally the method of choice. Recently, several procedures were described in the literature taking the advantages of the benefits of mass spectrometry. Both ESI and APCI sources as well as triple

As it was mentioned above the selection of appropriate mobile phase composition for determination of statins in biological fluids is quite challenging task which is even more complicated when detection and quantification of statins is performed using MS. Only few additives could enable good stability at pH range 4 to 5 as well as volatility and sensitive mass spectrometric response. Therefore, Di and co-workers [84] have pointed out the importance of the formic acid in lowering the pH of mobile phase. In this way pitavastatin was obtained in non-ionized form and a symmetrical peak shape was observed. The concentration of formic acid was optimized not only to maintain a symmetrical peak shape in the chromatographic system but also to render good ionization and fragmentation of pitavastatin in the MS/MS detector. An addition of 0.025% formic acid to the aqueous phase was found to be an important factor for acquiring the high sensitivity of another LC/MS/MS

Nováková and co-workers [83] have presented a nice example of optimization of the buffer pH and concentration in order to get the best signal to noise ratio of MS detector. Ammonium formate and ammonium acetate at pH 4.0 and 4.5 were tested at the concentration range 0.01 to 10 mM. The best response of atorvastatin and simvastatin was observed at 0.5 mM buffers. The concentrations higher than 5 mM significantly decreased the response of mass spectrometer. On the other hand, the concentrations lower than 0.5 mM were not sufficient to keep buffering capacity and thus had negative influence to the response of mass spectrometer. Ammonium acetate was preferred before ammonium formate because of better peak shapes. Finally, the optimized mobile phase composition was 70% of acetonitrile and 30% of ammonium acetate buffer 0.5 mM (pH 4.0). In most of bioanalytical methods isocratic elution has been utilized, still when more analytes with

Tandem mass spectrometry detection for identification and quantification of simvastatin and atorvastatin together with their metabolites and lacton/hidroxy acid interconversion forms was employed [83, 86]. All analytes were monitored using electrospray positive ionization (ESI+) mode and for all analytes protonated molecule [M+H]+ was the most intensive ion in mass spectra. Quantification of all analytes was performed using selected reaction monitoring (SRM) and two specific transitions were optimized for each molecule in order to increase selectivity and sensitivity of the method. In the paper published afterwards simvastatin in its lactone form was determined in ESI+ mode, while its hydroxy acid form

LC/MS/MS method developed by Apostolou and co-workers [75] consisted also of two periods combining both negative and positive ionization modes. The mass spectrometer

mode due to poor sensitivity of hydroxy acid form in positive ion

Rosuvastatin has a pyrimidine ring and a carboxylic group in its structure, hence it could be detected either in positive or negative ionization mode. However, the quantification of rosuvastatin in positive ionization mode is more common and was used for determination of rosuvastatin [80] and rosuvastatin together with its metabolites [74], respectively. In the both assays the major ion was protonated molecule [M+H]+ in full-scan mode and principal product ion was at *m/z* 482. Macwan and co-workers [74] have also reported two minor fragments at *m/z* 300 and *m/z* 272.

During the method development, Gao and co-workers [79] also attempted to optimize ESI conditions under positive ionization mode. However, the observed signal intensity was not sensitive enough for determination of expected rosuvastatin's concentrations, especially for low dosage administration. Low sensitivity of positive ionization mode could be explained by a number of fragment ions produced in the product ion spectrum of [M+H]+. In order to improve the sensitivity of the method, the negative ESI detection was taken into consideration. Under negative ESI mode, rosuvastatin produced abundant deprotonated molecule [M-H]- at m/z 480. In the product ion mass spectrum of [M-H]- , fewer fragment ions were formed compared with that of [M +H]+. Also, it was pointed out that negative ESI mode produced lower chemical background noise than positive. Comparing LOQ values obtained by these three methods, it can be observed that almost five times lower LOQ value for rosuvastatin was obtained using negative ESI detection.

Pitavastatin has similar structure to rosuvastatin. It contains alkaline nitrogen ion on the quinoline ring and a carboxylic group, therefore positive and negative ionization mode could be also employed. Both of ionization modes for determination of pitavastatin in human plasma and urine by LC/MS/MS method were applied [84]. The results showed that the response intensity of pitavastatin in negative mode was lower and furthermore the response was quite unstable. Pitavastatin was scanned under Q1 MS full-scan mode to determine the parent ion and under Q1/Q3 product ion scan mode to locate the most abundant production. The protonated molecular ion, [M+H]+, was the predominant ion in the Q1 spectrum and was used as the parent ion to obtain the product ion spectra. The most sensitive mass transition was from *m/z* 422.0 to 290.1, which was similar to the MS/MS

spectrum of pitavastatin reported in reference [88] and [93], while in the previously reported LC/MS/MS method the highest collision energy gave the most abundant product ion at *m/z* 318.0 [77].

A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 409

applications as they include analyte derivatization step prior to anaysis in order to obtain volatile derivatives of the drug molecule and therefore a complicate sample preparation

Simultaneous determination of lovastatin, simvastatin and pravastatin in plasma using GC with chemical ionization mass spectrometry has been described [70]. The analytes were isolated from plasma by SPE procedure which separated the lactone and acid forms of the drugs. The lactone forms were converted to the corresponding acid forms, which were subsequently derivatized by pentafluorobenzylation of the carboxyl group, and trimethylsilylation of the hydroxyl functions. The method has sufficient sensitivity for the analysis of clinical samples containing the drugs administered at therapeutic doses with recoveries between 79 and 90%. In another method, simvastatine and its acid form were

Far to our knowledge since 2001 no method for determination of statin drugs in biological samples using gas chromatography has been published due to imprecise and time consuming derivatization procedures which is an unavoidable step in analysis of statin

Since all HMG-CoA reductase inhibitors are given to the patients once daily, monitoring plasma concentrations over a period of 24 hours is necessary. In all published papers monitoring plasma concentration levels were performed at least over 24 hours, except in references [74, 89] were the blood samples were collected at various time points during a period of 12 hours after a single oral dose of rosuvastatin and atorvastatin, respectively. Also, in pharmacokinetic and bioavailability study of simvastatin in healthy volunteers and moderately hyperlipemic patients' drug plasma concentrations were monitored during 12 hours [96]. In the most of investigations pharmacokinetic parameters of statins were

Pharmacokinetic parameters of rosuvastatin have been investigated after single doses of 5, 10 and 20 mg [79]. The peak plasma levels obtained from this study were 8.32, 14.8 and 20.1 ng/mL, respectively. It was found that plasma exposure to rosuvastatin appeared increasing dose-proportionally and the plasma elimination half-lives were prolonged with increased doses. Not so many methods for determination of statins in human urine have been developed. The SPE-LC/MS/MS method was successfully applied to quantify the pitavastatin concentration in plasma and urine which were collected from Chinese volunteers [84]. The urinary excretion ratio of pitavastatin accounted for less than 0.6%, which suggested that pitavastatin was not excreted primarily by kidney. Quite similar data

Several above described bioanalytical assays have been used in bioequivalence studies of statin drugs. The pharmacokinetic parameters derived from drug plasma concentrations, including maximum plasma concentration, area under the plasma concentration-time curve from 0 h to the last measured data, area under the plasma concentration-time curve from 0 h

procedures.

derivatized with ferroceneboranic acid.

**3.4. Pharmacokinetic studies** 

molecules and the biggest disadvantage of using this technique.

investigated after only one pharmaceutical tablet dosage.

were obtained using LLE-LC/MS/MS method [88].

Recently, two LC/MS/MS methods have been developed for determination of pravastatin in human plasma [85, 94]. Both methods utilized ESI but in different modes. In the method developed by Martens and co-workers [85] the mass spectrometer was operated in the positive mode. The MS/MS detection was set up in MRM mode. The full scan mass spectra of pravastatin and its metabolites were scanned. The collision energy in Q2 produced different significant fragment ions. The MS/MS ion transitions selected for quantification purpose were *m/z* 442.2 to 269.1, 442.2 to 269.1 and *m/z* 424.3 to 183.0 for pravastatin, 3-OH metabolite and its lacton form. On the contrary, Polagani and co-workers [94] have found high sensitivity and stability using negative ionization mode. Deprotonated form of pravastatin, [M-H]- ion was the parent ion in the Q1 spectrum and was used as the precursor ion to obtain Q3 product ion spectra. The most sensitive mass transition was monitored from *m/z* 423.3 to 100.8.

Internal standards have been used in most of the assays leading to more corrected results. In some cases one of the statins has been used as internal standard [73, 75, 77, 84], while other works utilized internal standards of various structure, including hydrochlorothiazide [79], estrone [80], naproxen [90], gemfibrozil [91], pioglitazone [95] etc. The best internal standards for precise and accurate quantification in MS or tandem MS are stable-isotopelabeled standards. Only a few works employed deuterium labeled standards [74, 82, 83, 86]. In the case of atorvastatin, [d5] labeling usually occurs on the phenyl ring, which does not contain fluorine. [d3] labeling of simvastatin occurs on the side chain, while [d6] labeling of rosuvastatin occurs on isopropyl group attached to pyrimidin ring. In most of investigations only one compound was used as internal standard.

However, Mertens and co-workers [85] have used two different internal standards for quantification of fenofibric acid, pravastatin and its metabolites in human plasma by automated SPE-LC/DAD/MS/MS technique. To avoid the need for plasma dilution and two time-consuming analytical runs, the use of two internal standards was necessary as the concentration of fenofibric acid was too high and MS signal appeared saturated. Hence, the sulindac was selected for the quantification of fenofibric acid by UV-detector, while the triamcinolone was used for MS/MS quantification of pravastatin and its metabolites. As it was mentioned above, in the method developed by Zhang and co-workers [82], the LC/MS/MS data acquisition for simvastatin was conducted in positive ionization mode, whereas the data acquisition for simvastatin acid was conducted in negative ionization mode. Therefore, it was inevitable to use two internal standards deuterium labeled simvastatin and deuterium labeled simvastatin acid, respectively.

## **3.3. Gas chromatography**

Several GC/MS methods for determination of statins in biological samples have been reported [10]. Unfortunately, these methods are limited and not recommended for routine applications as they include analyte derivatization step prior to anaysis in order to obtain volatile derivatives of the drug molecule and therefore a complicate sample preparation procedures.

Simultaneous determination of lovastatin, simvastatin and pravastatin in plasma using GC with chemical ionization mass spectrometry has been described [70]. The analytes were isolated from plasma by SPE procedure which separated the lactone and acid forms of the drugs. The lactone forms were converted to the corresponding acid forms, which were subsequently derivatized by pentafluorobenzylation of the carboxyl group, and trimethylsilylation of the hydroxyl functions. The method has sufficient sensitivity for the analysis of clinical samples containing the drugs administered at therapeutic doses with recoveries between 79 and 90%. In another method, simvastatine and its acid form were derivatized with ferroceneboranic acid.

Far to our knowledge since 2001 no method for determination of statin drugs in biological samples using gas chromatography has been published due to imprecise and time consuming derivatization procedures which is an unavoidable step in analysis of statin molecules and the biggest disadvantage of using this technique.

## **3.4. Pharmacokinetic studies**

408 Chromatography – The Most Versatile Method of Chemical Analysis

only one compound was used as internal standard.

simvastatin and deuterium labeled simvastatin acid, respectively.

ion at *m/z* 318.0 [77].

from *m/z* 423.3 to 100.8.

**3.3. Gas chromatography** 

spectrum of pitavastatin reported in reference [88] and [93], while in the previously reported LC/MS/MS method the highest collision energy gave the most abundant product

Recently, two LC/MS/MS methods have been developed for determination of pravastatin in human plasma [85, 94]. Both methods utilized ESI but in different modes. In the method developed by Martens and co-workers [85] the mass spectrometer was operated in the positive mode. The MS/MS detection was set up in MRM mode. The full scan mass spectra of pravastatin and its metabolites were scanned. The collision energy in Q2 produced different significant fragment ions. The MS/MS ion transitions selected for quantification purpose were *m/z* 442.2 to 269.1, 442.2 to 269.1 and *m/z* 424.3 to 183.0 for pravastatin, 3-OH metabolite and its lacton form. On the contrary, Polagani and co-workers [94] have found high sensitivity and stability using negative ionization mode. Deprotonated form of pravastatin, [M-H]- ion was the parent ion in the Q1 spectrum and was used as the precursor ion to obtain Q3 product ion spectra. The most sensitive mass transition was monitored

Internal standards have been used in most of the assays leading to more corrected results. In some cases one of the statins has been used as internal standard [73, 75, 77, 84], while other works utilized internal standards of various structure, including hydrochlorothiazide [79], estrone [80], naproxen [90], gemfibrozil [91], pioglitazone [95] etc. The best internal standards for precise and accurate quantification in MS or tandem MS are stable-isotopelabeled standards. Only a few works employed deuterium labeled standards [74, 82, 83, 86]. In the case of atorvastatin, [d5] labeling usually occurs on the phenyl ring, which does not contain fluorine. [d3] labeling of simvastatin occurs on the side chain, while [d6] labeling of rosuvastatin occurs on isopropyl group attached to pyrimidin ring. In most of investigations

However, Mertens and co-workers [85] have used two different internal standards for quantification of fenofibric acid, pravastatin and its metabolites in human plasma by automated SPE-LC/DAD/MS/MS technique. To avoid the need for plasma dilution and two time-consuming analytical runs, the use of two internal standards was necessary as the concentration of fenofibric acid was too high and MS signal appeared saturated. Hence, the sulindac was selected for the quantification of fenofibric acid by UV-detector, while the triamcinolone was used for MS/MS quantification of pravastatin and its metabolites. As it was mentioned above, in the method developed by Zhang and co-workers [82], the LC/MS/MS data acquisition for simvastatin was conducted in positive ionization mode, whereas the data acquisition for simvastatin acid was conducted in negative ionization mode. Therefore, it was inevitable to use two internal standards deuterium labeled

Several GC/MS methods for determination of statins in biological samples have been reported [10]. Unfortunately, these methods are limited and not recommended for routine Since all HMG-CoA reductase inhibitors are given to the patients once daily, monitoring plasma concentrations over a period of 24 hours is necessary. In all published papers monitoring plasma concentration levels were performed at least over 24 hours, except in references [74, 89] were the blood samples were collected at various time points during a period of 12 hours after a single oral dose of rosuvastatin and atorvastatin, respectively. Also, in pharmacokinetic and bioavailability study of simvastatin in healthy volunteers and moderately hyperlipemic patients' drug plasma concentrations were monitored during 12 hours [96]. In the most of investigations pharmacokinetic parameters of statins were investigated after only one pharmaceutical tablet dosage.

Pharmacokinetic parameters of rosuvastatin have been investigated after single doses of 5, 10 and 20 mg [79]. The peak plasma levels obtained from this study were 8.32, 14.8 and 20.1 ng/mL, respectively. It was found that plasma exposure to rosuvastatin appeared increasing dose-proportionally and the plasma elimination half-lives were prolonged with increased doses. Not so many methods for determination of statins in human urine have been developed. The SPE-LC/MS/MS method was successfully applied to quantify the pitavastatin concentration in plasma and urine which were collected from Chinese volunteers [84]. The urinary excretion ratio of pitavastatin accounted for less than 0.6%, which suggested that pitavastatin was not excreted primarily by kidney. Quite similar data were obtained using LLE-LC/MS/MS method [88].

Several above described bioanalytical assays have been used in bioequivalence studies of statin drugs. The pharmacokinetic parameters derived from drug plasma concentrations, including maximum plasma concentration, area under the plasma concentration-time curve from 0 h to the last measured data, area under the plasma concentration-time curve from 0 h


A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 411

to the infinity, the time to reach peak concentration, the apparent elimination rate constant, showed that there was no statistically significant difference between two investigated

Not so many chromatographic methods have been developed for the quantification of HMG-CoA reductase inhibitors in combination with their metabolites. They undergo quite extensive first-pass metabolism during witch active and inactive metabolites are produced. The actual plasma concentrations of both parent compounds and metabolites are of major interest in pharmacokinetics studies. Therefore, analytical methods for simultaneous determination of statins and their metabolites are quite valuable. Although simultaneous determination of statins and their metabolites was considered being difficult owing to the different polarities of the analytes, several methods have been

Recently, Apostolou and co-workers [75] published fast and fully automated LLE-LC/MS/MS method, while Zhang and co-workers [82] presented a high-throughput saltingout assisted LLE-LC/MS/MS method for simvastatin in lactone and acid form. Both of methods were very fast with analytical runs less than two min and fairly sensitive with LOQ values around 0.1 ng/mL. Nováková and co-workers [83] have developed fast selective and reliable SPE-UPLC/MS/MS method for simultaneous determination of simvastatin and atorvastatin as well as their active and inactive metabolites. The main advantage of the method was applicability of the method for determination of two clinically widely used statins using one sample preparation procedure and one chromatographic run, while the main limitation of study was slightly higher LOQ value obtained for simvastatin in open-

More recently Vlčková and co-workers [86] have presented a new MEPS-UPLC method for determination of atorvastatin and its metabolites, faster and more sensitive comparing to previously published ones. A simple, fast and reproducible method for determination of rosuvastatin and metabolites in human plasma has been described [74]. The major advantages of the method were the requirement for small plasma volume and simple sample preparation procedure, protein precipitation. The major limitation of method was its inability to determine *N*-desmethyl rosuvastatin in the patient samples although its LOQ was quite low, 0.5 ng/mL. The patients included in the study took a single dose of rosuvastatin at 20 mg. *N*-desmethyl rosuvastatin is a minor metabolite that is present in much lower concentrations than rosuvastatin. Therefore, the authors anticipate that the methods should be sensitive enough to measure its concentration in patients receiving

A sensitive and accurate procedure based on solid-phase extraction coupled at-line to a LC/MS/MS for determination of pravastatin and its two metabolites in human plasma has been presented [85]. Optimized and validated LLE-LC/MS/MS method for determination of pitavastatin and its lacton form in human plasma as well as in urine is described [88].

pharmaceutical formulations [72, 73].

published.

ring hydroxy acid form.

rosuvastatin on a routine basis.

LOV-lovastatin, SIM-simvastatin, PRA-pravastatin, FLU-fluvastatin, ATO-atorvastatin, ROS-rosuvastatin, PITpitavastatin, VAL-valsartan, FFA-fenofibric acid, CLT-chlorthalidone, ACN-acetonitrile, IS-internal standard

**Table 3.** Analytical methods for the determination of statins in biological samples

to the infinity, the time to reach peak concentration, the apparent elimination rate constant, showed that there was no statistically significant difference between two investigated pharmaceutical formulations [72, 73].

410 Chromatography – The Most Versatile Method of Chemical Analysis

**Stationary phase Mobile phase Separation** 

gradient elution A: 0.1% glacial acetic acid in 10 % methanol in water B: 40% methanol in ACN

gradient elution

gradient elution A: ACN

(75:25:0.05)

B: 0.5 mM ammonium acetate buffer pH 4.0

ACN:methanol:5 mM ammonium acetate buffer pH 4.5 (30:30:40)

B: 0.5 mM ammonium acetate pH 4.0

ammonium formate, 0.01% formic acid, pH 4.1

methanol:0.2% acetic acid in water (70:30)

gradient elution A: ACN

gradient elution A: 10 mM ammonium formate, 0.01% formic acid

B: ACN, 10 mM

methanol:water:formic acid

pH 4.1

ACN:5 mM ammonium acetate pH 4.5 (82:18)

A: ACN, 0.01% formic acid, 10 mM ammonium formate B: 0.01% formic acid, 10 mM ammonium formate

methanol:water (75:25), pH 6.0 with ammonia

**technique and Detection** 

HPLC ESI+ MS/MS MRM

HPLC APCI/ESI- / ESI+ MS/MS MRM

HPLC UV 229, 254, 236 nm FD 254, 378 nm

HPLC ESI-MS/MS MRM

UPLC ESI+ MS/MS SRM

HPLC ESI+ MS/MS SRM

HPLC ESI+ MS/MS MRM

UPLC ESI+ MS/MS SRM

UPLC UV 220 nm

HPLC ESI+ MS/MS MRM

**LOQ Ref.** 

74

75

76

79

83

84

85

86

87

88

0.1– 0.5 ng/mL

0.1 ng/mL

UV: 31-85 μg/mL FD: 10-20 μg/mL

0.02 ng/mL

0.09– 4.38 nM

0.08 ng/mL

0.05– 0.5 ng/mL

0.08- 0.66 nM

20-110 μg/mL

1 ng/mL

**Sample preparation** 

plasma Agilent Zorbax-SB

plasma YMC ODS-A (50 x 4.0 mm)

plasma Waters Atlantis

plasma Agilent Zorbax

serum Waters Acquity

μm)

plasma, urine Shimadzu Shim-

plasma Phenomenex

serum Waters BEH C18

plasma Waters Acquity

μm)

μm)

plasma, urine Thermo BDS

μm)

Phenyl, Rapid Resolution HT (100 x 2.1 mm, 3.5 μm)

dC18 (100 x 3.9, 3

XDB-C18 (150 x 4.6 mm, 5 μm)

UPLCTM BEH C18 (100 x 2.1 mm, 1.7

pak VP-ODS (150 x 4.6 mm, 5 μm)

Synergi Max-RP (150 x 2 mm, 4 μm)

(100 x 2.1 mm, 1.7

UPLCTM BEH C18 (50 x 2.1 mm, 1.7

Hypersil C8 (50 x 2.1 mm, 3 μm)

**Table 3.** Analytical methods for the determination of statins in biological samples

LOV-lovastatin, SIM-simvastatin, PRA-pravastatin, FLU-fluvastatin, ATO-atorvastatin, ROS-rosuvastatin, PITpitavastatin, VAL-valsartan, FFA-fenofibric acid, CLT-chlorthalidone, ACN-acetonitrile, IS-internal standard

**Analytes Matrix /** 

ROS + metabolites, IS=deuterium labeled

SIM, SIM acid IS= LOV, LOV acid

CLT

ROS

PIT IS=ROS

CLT

FLU, VAL + metabolite,

IS=candesartan cilexetil

IS=hydrochlorothiazide

SIM, SIM-acid, ATO + metabolites IS=deuterium labeled

PRA + metabolites, FFA IS=triamcinolone

ATO + metabolites IS=deuterium labeled

FLU, VAL + metabolite,

IS=candesartan cilexetil

PIT, PIT-lacton IS=racemic i-prolact Not so many chromatographic methods have been developed for the quantification of HMG-CoA reductase inhibitors in combination with their metabolites. They undergo quite extensive first-pass metabolism during witch active and inactive metabolites are produced. The actual plasma concentrations of both parent compounds and metabolites are of major interest in pharmacokinetics studies. Therefore, analytical methods for simultaneous determination of statins and their metabolites are quite valuable. Although simultaneous determination of statins and their metabolites was considered being difficult owing to the different polarities of the analytes, several methods have been published.

Recently, Apostolou and co-workers [75] published fast and fully automated LLE-LC/MS/MS method, while Zhang and co-workers [82] presented a high-throughput saltingout assisted LLE-LC/MS/MS method for simvastatin in lactone and acid form. Both of methods were very fast with analytical runs less than two min and fairly sensitive with LOQ values around 0.1 ng/mL. Nováková and co-workers [83] have developed fast selective and reliable SPE-UPLC/MS/MS method for simultaneous determination of simvastatin and atorvastatin as well as their active and inactive metabolites. The main advantage of the method was applicability of the method for determination of two clinically widely used statins using one sample preparation procedure and one chromatographic run, while the main limitation of study was slightly higher LOQ value obtained for simvastatin in openring hydroxy acid form.

More recently Vlčková and co-workers [86] have presented a new MEPS-UPLC method for determination of atorvastatin and its metabolites, faster and more sensitive comparing to previously published ones. A simple, fast and reproducible method for determination of rosuvastatin and metabolites in human plasma has been described [74]. The major advantages of the method were the requirement for small plasma volume and simple sample preparation procedure, protein precipitation. The major limitation of method was its inability to determine *N*-desmethyl rosuvastatin in the patient samples although its LOQ was quite low, 0.5 ng/mL. The patients included in the study took a single dose of rosuvastatin at 20 mg. *N*-desmethyl rosuvastatin is a minor metabolite that is present in much lower concentrations than rosuvastatin. Therefore, the authors anticipate that the methods should be sensitive enough to measure its concentration in patients receiving rosuvastatin on a routine basis.

A sensitive and accurate procedure based on solid-phase extraction coupled at-line to a LC/MS/MS for determination of pravastatin and its two metabolites in human plasma has been presented [85]. Optimized and validated LLE-LC/MS/MS method for determination of pitavastatin and its lacton form in human plasma as well as in urine is described [88].

Furthermore, a LC/MS/MS method for separation of fluvastatin from its *threo* isomers metabolites to support a bioequivalence study has been developed [97].

A Review of Current Trends and Advances in Analytical Methods for Determination of Statins: Chromatography and Capillary Electrophoresis 413

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

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

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

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

0.01 ng/mL, 0.2 ng/mL and 20 ng/mL, respectively.

impurities in bulk drug [108].

analysis of lovastatin.

are scarce.

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] and losartan, atenolol, acetylsalicylic acid [99].

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 drugs.

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].
