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HPLC methods for the determination of simvastatin and atorvastatin
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Trends in Analytical Chemistry, Vol. 27, No. 4, 2008
HPLC methods for the determination of simvastatin and atorvastatin Lucie Nova´kova´, Dalibor Sˇatı´nsky´, Petr Solich We review high-performance liquid chromatography (HPLC) methods for the determination of two major statins used in clinical treatment – simvastatin and atorvastatin – in various fields of application, including bio-analytical assays, pharmaceutical assays and environmental applications. Statin molecules are known to be susceptible to interconversion of the lactone and acidic forms, so it is necessary to consider this phenomenon during method development. We highlight liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) methods, as they have become a method of choice in bio-analytical and environmental applications. We compare the methods from the point of view of sensitivity. We discuss selection of the precursor ion for performing selected reaction monitoring (SRM) in MS detection and sample preparation. ª 2008 Elsevier Ltd. All rights reserved. Keywords: Atorvastatin; Bio-analytical method; Environmental analysis; High-performance liquid chromatography; HPLC; Interconversion; Pharmaceutical analysis; Simvastatin; Tandem mass spectrometry Abbreviations: [13CD3], Stable-isotope labeling using 13C and three atoms of deuterium; [d5], Stable isotope labeling using five atoms of deuterium; 2-OH-AT, 2-hydroxyatorvastatin; AcAc, Acetic acid; ACN, Acetonitrile, AmAc, Ammonium acetate; AT, Atorvastatin; AT-L, Atorvastatin lactone; DFAT, Desfluoro-atorvastatin; DSAT, Diastereomer-atorvastatin; ESI, Electrospray ionization; Et-Ac, Ethylacetate; FAc, Formic acid; FD, Fluorescence detection; GC, Gas chromatography; HLB, Hydrophilic lipophilic balance; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HPLC, High-performance liquid chromatography; IS, Internal standard; LDL, Low-density lipoprotein; LLCE, Liquid-liquid cartridge extraction; LLE, Liquid-liquid extraction; LOD, Limit of detection; LOQ, Limit of quantitation; LOV, Lovastatin; LOVA, Lovastatin acid; M, mol/l; MeOH, Methanol; Me- SV, Methyl-simvastatin; MEV, Mevastatin; MP, Mobile phase; MS, Mass spectrometry; MS/MS, Tandem MS; MTBE, Methyl-tert-butyl-ether; ODS, Octadecylsilica, C18; o-OH-AT, o-hydroxyatorvastatin; p-OH-AT, p-hydroxyatorvastatin; PRA, Pravastatin; QC, Quality control; ROS, Rosuvastatin; SDS, Sodium dodecyl-sulphate; SIM, Selected ion monitoring; SPE, Solid-phase extraction; SRM, Selective reaction monitoring; SV, Simvastatin; SVA, Simvastatin acid; THF, Tetrahydrofuran; TIS, Turbo ionspray; ulv, ultra-low volume; UV, Ultraviolet
1. Introduction Lucie Nova´kova´*, Dalibor Sˇatı´nsky´, Petr Solich Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovske´ho 1203, 500 05, Hradec Kra´love´, Czech Republic
*
Corresponding author. Tel.: +420 495067345; Fax: +420 495067164; E-mail: [email protected]
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Statins include natural (lovastatin), semisynthetic (simvastatin, and pravastatin) and synthetic compounds (fluvastatin, atorvastatin, cerivastatin, rosuvastatin and pitavastatin) and are potent, specific and competitive inhibitors of 3-hydroxy-3methlyglutaryl coenzyme A (HMG-CoA) reductase. They are highly effective in reducing total cholesterol and low-density lipoprotein (LDL) cholesterol levels in the human body. HMG-CoA reductase is the key enzyme that catalyzes the conversion of HMG-CoA to mevalonate, which is an early ratelimiting step in the biosynthetic pathway of cholesterol (Fig. 1). High-plasma LDL cholesterol is a risk factor of cardiovascu-
lar diseases, such as atherosclerosis, which is characterized by deposition of cholesterol on the arterial wall [1–3]. Atherosclerosis of the coronary and peripheral vasculature is the leading cause of death worldwide. Statistics show that 38–42% of deaths are related to cardiovascular diseases in Western and other developed countries [4]. Lowering cholesterol levels can arrest or reverse atherosclerosis in all vascular beds and it can significantly decrease the morbidity and the mortality associated with atherosclerosis. The main methods of treating hyperlipidemia (or hypercholesterolemia) are dietary and lifestyle changes and the administration of hypolipidemic drugs [5]. Statins are commonly used to treat several forms of hypercholesterol-
0165-9936/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.01.013
Trends in Analytical Chemistry, Vol. 27, No. 4, 2008
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Figure 1. Biosynthesis of cholesterol. Cholesterol is synthesized from acetyl coenzyme A. The synthesis of mevalonate, mediated by HMG-CoA reductase is the rate-limiting step, which regulates the cholesterol synthesis. It is the step where the statin molecule competitively effects the synthesis of cholesterol.
emia.They have potent cholesterol-lowering effects and they could reduce morbidity and mortality associated with coronary heart disease significantly, as proved by many clinical trials [2,6–8]. However, some statins exhibit a number of adverse effects, such as myopathy or rhabdomyolysis, so it is useful to monitor the levels of statins in biological materials in order to establish an appropriate dosage scheme, which would minimize adverse effects and keep the cholesterol-lowering effect [2]. Simvastatin and atorvastatin are the two most commonly occurring drugs in commercially available pharmaceutical formulations used for the clinical treatment of hypercholesterolemia. Their structures can be seen in Fig. 2.
2. Chemistry and pharmacokinetics The first statin molecule – mevastatin – was discovered by Endo et al. in 1976 as a fungal product extracted from Penicillium citrinum [9]. Lovastatin, simvastatin and pravastatin are also derivatives of fungal products. Simvastatin and pravastatin are nowadays produced semi-synthetically from lovastatin and mevastatin. However, fluvastatin is a completely synthetic statin with a very different structure from statins derived from fungal products. It is a mevalonolactone derivative with a fluorophenyl-substituted indole ring. Other synthetic statins (cerivastatin, atorvastatin, rosuvastatin and pitavastatin) have similar structures with fluorophenyl groups. All of totally synthetic statins have open-ring
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Trends in Analytical Chemistry, Vol. 27, No. 4, 2008 OH O CH3
H3C
O
O
H
H3C
O
CH3
H3C
Simvastatin (lactone form)
HO COOH OH
CH3
F N
CH3 H N O
Atorvastatin (acid form)
Figure 2. Chemical structures of simvastatin and atorvastatin.
acid forms [2,9]. All statins are absorbed rapidly following administration, reaching peak plasma concentration within 4 hours. Statins exist in two forms, lactone and open-ring hydroxy acid [10,11]. In vivo, the hydroxy acid forms are the active drugs that lower plasma cholesterol while the lactone forms are inactive (prodrug). The lactone form of statin can be absorbed from the gastrointestinal tract and transformed into the active drugs in liver and non-hepatic tissues [11]. Depending upon chemical structure, statins have different affinities for HMG-CoA reductase, which determines their pharmacological effects and different pharmacokinetic properties (e.g., tissue distribution, metabolic stability, enzymes and transporters involved in their metabolism) [2]. We will provide further information on simvastatin and atorvastatin, the most widely used statins in clinical treatment – Fig. 2. Simvastatin is a prodrug, which is administered as an inactive lactone form. The lactone is absorbed from gastrointestinal tract and hydrolyzed to the active b-hydroxy acid form in the liver. Simvastatin and its hydroxy acid are extensively (95%) bound to plasma proteins. The substance undergoes extensive first-pass metabolism in the liver and is mainly excreted in the bile. About 85% of administered dose has been recovered from the feces as metabolite and about 10–15% from urine, mainly as inactive forms [12,13]. Atorvastatin is administered in the open-ring hydroxy acid form – the active form. It is absorbed from the 354
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gastrointestinal tract and it undergoes extensive firstpass metabolism in the liver. Liver metabolism produces two active hydroxy metabolites – ortho-hydroxyatorvastatin and para-hydroxyatorvastatin – and three inactive metabolites corresponding to the lactone form. More than 98% is bound to plasma proteins. It is excreted mainly in feces via the bile, with a smaller proportion excreted in the urine. About 70% of the total plasma HMG-CoA activity is attributed to active metabolites of atorvastatin, even if their concentrations are very low [12–14]. As apparent from the information above, the levels of statins in biological fluids are very low, probably because only about 5% of dosed statin reaches the systemic circulation. For atorvastatin and simvastatin, the plasma concentrations are typically at ng/ml levels. The active metabolites of atorvastatin are present at plasma concentration corresponding to pg/ml levels [13], the typical concentration range being 0.1–20 ng/ ml.
3. Interconversion of lactone and hydroxy acid forms A number of classes of drugs could potentially undergo interconversion, which may occur during any of the numerous steps of the bio-analytical method: in the biological matrix before collecting aliquots of samples for the analysis; during extraction; during evaporation to dryness or reconstitution; in the solution in the injection vial; and, in the case of MS, in the ion source as well. There are several categories of drugs that could undergo interconversion. HMG-CoA reductase inhibitors are typical examples of such a class, where the interconversion occurs between lactone and open-ring hydroxy acid [10,11]. Another category could be conversion between samples that contain a carboxylic acid and its acylglucuronide [15]. Samples that contain a thiol group and its disulfide may also interconvert to each other [16]. Minimizing the interconversion will depend on the conditions during the bio-analytical procedure, pH being one of the most important. HMG-CoA reductase inhibitor simvastatin is administered in its lactone form, although the active form is open-ring hydroxy acid. Both forms are found in postdose samples. On the other hand, atorvastatin is administered as the open-ring hydroxy acid, but the post-dose samples contain both acid and lactonized forms [10,11,17,18]. For the samples of hydroxy acid chemical structure and the corresponding lactone forms, it is important to maintain pH between 4 and 5 in order to minimize
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interconversion. Increasing the pH above 6 facilitates the conversion of lactone to acid (in the ionized form); by contrast, lowering pH facilitates the conversion of acid to lactone or lactone to acid (in the non-ionized form). Most assays utilize pH around 4.5 (see Tables 1 and 2). It is important to optimize the method in order to minimize such interconversion. Unfortunately, even optimal conditions may not totally prevent interconversion. It is thus essential to design the composition of the calibration standards and quality control (QC) samples in terms of the ratio of the concentration of one analyte to that of the other so that the accuracy and precision obtained for QC samples realistically reflect accuracy and precision that will be obtained for the expected samples. Ideally, the composition of the QC samples should be identical to that of real samples. This is difficult to establish at the early stage of assay, where the composition of real samples is not known and will probably change from sample to sample [10]. It is recommended that the example of methoddevelopment design should include QC samples prepared in real biological material containing only the lactone form, only the hydroxy acid form, and both in varying ratios of lactone to hydroxy acid (1:1; 1:10; 10:1; 1:3; and, 3:1) in order to get good method accuracy and precision. Such QC samples should be prepared at various concentration levels inside the range of calibration curve [10]. In this study, the influence of pH (analytical conditions at pH 4.2, 7.3 and 1.8) on interconversion of analytes was described in detail. Under the conditions that showed practically no interconversion, the results for all QC samples gave excellent values for accuracy (up to 8.6% RSD) and precision (up to 8.7% RSD) with no influence of the ratio of both analytes. However, in another experimental design, the results for accuracy and precision were strongly influenced by this ratio, except for 1:1, which was the same ratio as in the calibration standards. Thus, under the conditions that allow interconversion between analytes, a method validated for the quantitation of the two analytes using calibration standards with the ratio of analyte concentration of 1:1 and QC samples with the same 1:1 ratio can be used for accurate measurements of the analytes in real samples only if such samples also contain the two analytes in the ratio 1:1. Therefore, to obtain an accurate indication of the performance of the method for all real samples, it is necessary to use QC samples that cover the entire spectrum of composition of real samples. To summarize, in developing a method for the quantitation of two analytes that can undergo interconversion, the first step is to select the conditions that will eliminate or minimize interconversion. The second step is judicious selection of the composition of the QC samples and the composition of calibration standards, which should cover the spectrum of the composition of real samples [10].
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4. Analytical methods The determination of drugs is a multi-disciplinary task. During the manufacturing process of a drug substance and drug formulation, there is a need for QC analytical methods to include all known and unknown impurities. Bio-analytical methods are necessary for clinical trials, therapeutic drug monitoring and individual dosagescheme adjustment. Recently, there has also been great interest in monitoring pharmaceutical residues in the environment, so environmentally-focused analytical methods are also necessary. Determination of drugs in biological materials is an important step in drug discovery and drug development, as it provides pharmacokinetic information, and is necessary when the treatment-dose schedule and safety margins need to be established. High-performance liquid chromatography (HPLC) together with various types of detection – UV (ultraviolet), FD (fluorescence detection) and MS – has become the method of choice for bio-analytical method development. Gas chromatography (GC) is somewhat suppressed because of the need for a derivatization step prior to analysis in order to obtain volatile derivatives of the drug molecule, which is often not volatile in the case of pharmaceuticals. As expected from the different structures of simvastatin and atorvastatin, analytical methods for their quantitative determination were developed individually. Because of the structural properties, there are not many analytical methods that determine these two compounds together in one analytical run or even in combination with other statin molecules. This is also probably because statins are not used with other statins simultaneously during treatment of hyperlipidemic patients. There are only two works that describe the determination of a number of statin structures together in one analytical run: an environmental application – where simvastatin, atorvastatin, lovastatin and pravastatin (internal standard (IS) = mevastatin) were determined in aqueous samples [19]; and, a pharmaceutical application – where atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin were determined together for QC of pharmaceutical formulations [20]. Theophylline was employed as IS for quantitation. The analytical run took about 40 min. There have been two reviews on analytical methods for the determination of HMG-CoA reductase inhibitors. The first, published in 2003, referred to lovastatin, simvastatin, pravastatin, fluvastatin and atorvastatin [12]. HPLC and GC methods were discussed. Generally, fluorescence and UV detection were applied together with HPLC. Only two methods for the determination of atorvastatin were available at that time. The second http://www.elsevier.com/locate/trac
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Table 1. HPLC analytical methods for the determination of simvastatin Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor ions notes
Time [min]
Validation data LOQ/LOD
Ref.
SV, SVA LOV, LOVA PRA
Pu-Ehr tea SPE
Luna C18 (4.6 · 250 mm, 5 lm)
ACN, water, AcAc (70:30:0.5)
ESI+ MS-MS SRM
Not stated
15
Not given stability and interconversion study
[11]
SV, AT LOV PRA IS = MEV
Aqueous samples SPE
Genesis C18 (2.1 · 50 mm, 3 lm)
Gradient elution A: ACN + 2 mM MeA + 0.1% AcAc B: water + 2 mM MeA + 0.1% AcAc
ESI+ MS-MS SRM
[M + CH3NH3]+ [M + H]+ [M + CH3NH3]+ [M + CH3NH3]+
5.5
LOD = 0.1–1.0 ng/l LOD = 0.1–1.2 ng/l LOD = 0.1–1.2 ng/l LOD = 1.0–15.4 ng/l
[19]
SV AT, LOV PRA, ROS IS = theophylline
Pharmaceutical formulation metabolism study in vitro extraction MeOH
Interstil ODS 3V (4.6 · 250 mm, 5 lm)
0.01 M AmAc pH 5.0 ACN MeOH
UV 237 nm
–
40
LOQ = 0.1 lg/ml
[20]
SV, SVA IS = LOV, LOVA
Human plasma SPE
Capcell Pak C18 (4.6 · 150 mm, 5 lm)
ACN-water (80:20)
FD 360 nm 430 nm
Derivatization by 1-bromoacetylpyrene
35
LOQ = 0.1 ng/ml
[21]
SV, SVA No IS
Human plasma LLE
ODS Hypersil (4.6 · 250 mm, 5 lm)
0.025 M sodium dihydrogenphosphate pH4.5 ACN (35:65)
UV 238 nm
–
10
LOD = 15 ng/ml
[22]
SV IS = LOV
Human plasma LLE
Capcell Pak C18 UG 120U (1.5 · 250 mm, 5 lm)
ACN 20 mM potassium phosphate buffer pH 5.6 (65:35)
–
30
LOQ = 0.5 ng/l
[23]
SV and impurities (SVA, LOV Me-SV, dimmer acetate ester anhydro-SV IS = propyphenazon
Tablets extraction MP
X-Terra (4.6 · 50 mm, 3.5 lm)
Microemulsion: 0.9% diisopropylether 1.7% SDS 7.0% co-surfactant 90% 25 mM di-sodium phosphate pH 7.0
–
25
LOD = 5 ng/ml LOQ = 10 ng/ml (SV and SVA)
[24]
UV 238 nm
UV 238 nm
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Determined substances
Table 1 (continued) Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor ions notes
Time [min]
Validation data LOQ /LOD
Ref.
SV, LOV
Standard solutions
No LC
No LC
ESI+MSMS SRM
[M+H]+its fragmentations using MS
–
Not stated fragmentation study
[25]
SV, SVA
Standard solutions
C18 (3.9 · 50 mm, 5 lm)
ACN, 3.0 mM FAc (70:25)
ESI+ MS-MS SRM
[M + H]+ [M + NH4]+
2.5
Chromatographic and mass resolution study
[26]
SV + impurities No IS
SV substance tablets Extraction ACN/FAc
Zorbax C8 (4.6 · 150 mm, 3.5 lm)
Gradient elution A: 0.085 phosphoric acid B:ACN A: 0.001% FAc in water B: 0.001% FAc in CAN
DAD ESI+ MS-MS
45
Identification of impurities
[27]
SV, SVA IS = LOV, LOVA
Human plasma SPE
Discovery C18 (4.6 · 50 mm, 5 lm)
ACN, MeOH, AmAc 0.1 M (62:10:28)
ESI+/ MS-MS SRM
[M + CH3CN + Na]+ [M H]
4.5
LOQ = 0.03 ng/ml SV LOQ = 0.02 ng/ml SVA
[28]
SV, SVA IS = LOV, LOVA
Human plasma on-line SPE
C18 (3.9 · 50 mm, 5 lm)
ACN, 3.0 mM FAc (70:25)
ESI+ MS-MS SRM
[M + H]+
2.5
LOQ = 0.5 ng/ml SV
[29]
SV IS = LOV
Human plasma LLE
Shim-pack ODS (4.6 · 150 mm, 5 lm)
MeOH–water (9:1)
ESI+ MS SV
[M + Na]+
5
LOD = 0.05 ng/ml
[30]
SV, SVA IS = deuterium labeled
Eects of mobile phase on ionization/ fragmentation plasma LLCE
Kromasil C18 (2.0 · 50 mm, 4 lm)
ACN-buffer 2 mM pH 4.5 (70:30) buffers: AmAc, hydrazine acetate, alkylammonium acetates
ESI+ MS-MS SRM
[M H] SVA various SV: [M + H]+ [M + Na]+ [M + K]+ [M + NH4]+ [M + RNH3]+ R = alkyl
3.5
LOQ = 50 pg/ml
[31]
SV, SVA IS = stable isotope labeled
Human plasma LLCE
Kromasil C18 (2.0 · 50 mm, 5 lm)
ACN AmAc pH 4.5 by FAc (75:25)
TIS + MS-MS SRM
[M + H] SV [M H] + SVA
3.5
LOQ = 50 pg/ml
[32]
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Determined substances
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[34] LOQ = 0.05 ng/ml Kromasil C18 (2.0 · 50 mm, 5 lm) Human plasma LLE SV, SVA IS = stable isotope labeled
ACN 1 mM methyl ammonium acetate pH 4.5 (75:25)
TIS + MS-MS SRM
[M + H] SV [M H] + SVA
3.5
[33] LOQ = 0.05 ng/ml 3.0 [M H] SVA [M + CH3NH3]+ SV TIS+ MS-MS SRM Synergie Max RP (2.0 · 50 mm, 4 lm) Human plasma ulv-SPE
ACN 1 mM methyl ammonium acetate pH 4.5 (80:20)
Precursor ions notes
SV, SVA IS = deuterium labeled
Table 1 (continued)
Stationary phase analytical column Matrix sample preparation Determined substances
Mobile phase
Detection
Time [min]
Ref.
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Validation data LOQ /LOD
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work [13] referred to some MS bio-analytical methods for the determination of statins. Fig. 3 shows the trend in developing new methods for the determination of simvastatin and atorvastatin. Simvastatin has been monitored since the early 1990s, while, due to its later synthesis, atorvastatin has been monitored since 1999. Since 2003, the number of newly developed methods has been increasing for both analytes. 4.1. Simvastatin LC-MS/MS (LC coupled to tandem MS) is nowadays the method of choice for the determination of simvastatin and its hydroxy acid (Table 1). While, in 2003, there was the same number of GC methods (3), HPLC-UV or FD (3) and LC-MS (3) [12], newly developed methods employed MS/MS. Only simvastatin acid (open-acid form, which arises from interconversion during analytical procedure or in the human body as an active form of a prodrug) was monitored together with simvastatin; no other metabolites were described. Whereas the HPLC-FD method [21] using 1-bromoacetylpyrene for the derivatization could be very convenient for bio-analytical purposes with its LOQ of 0.1 ng/ml, the HPLC-UV assay has borderline sensitivity for the quantitation of simvastatin in biological materials because of the poor UV-absorption properties of simvastatin molecule, giving an LOD of 15 ng/ml [22]. A more sensitive HPLC-UV assay for the determination of simvastatin in human plasma was developed later with an LOQ of 0.5 ng/ml [23]. However, lower sensitivity is no problem for quantitation in pharmaceutical preparations. Such a method was developed by Malenovic et al. [24] utilizing a microemulsion mobile phase for quantitation of simvastatin and its six impurities (simvastatin acid, lovastatin, methyl-simvastatin, dimmer of simvastatin, acetate ester of simvastatin and anhydro-simvastatin) in tablets or the one previously cited [20]. HPLC-UV and FD analytical methods utilized C18 analytical columns for the retention/separation of simvastatin/simvastatin acid together with mobile phase containing acetonitrile in combination with phosphate buffer (to maintain pH around 4–5) or with water in an older method [21]. X-terra hybrid stationary phase was employed in one case together with application of a microemulsion mobile phase [24]. Isocratic elution was applied in most analyses. Detection was performed at 238 nm in all cases [22–24]. A fluorescence method utilized 360 nm and 430 nm [21]. Analytical run time was usually very long, starting from 10 min [22] going up to 35 min using FD [21]. Two bio-analytical methods also determined simvastatin acid next to the lactone form [21,22], as did a pharmaceutical QC method, where simvastatin acid was determined among six impurities using IS propyphenazon [24]. Lovastatin was often employed as an IS because of its unique structural
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Determined substances
Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor ions (notes)
Time [min]
Validation data LOQ /LOD
Ref.
AT, SV LOV, PRA IS = MEV
Aqueous samples SPE
Genesis C18 (2.1 · 50 mm, 3 lm)
Gradient elution A: ACN + 2 mM MeA + 0.1% AcAc B: water + 2 mM MeA + 0.1% AcAc
ESI + MS-MS SRM
[M + CH3NH3]+ [M + H]+ [M + CH3NH3]+ [M + CH3NH3]+
5.5
LOD = 0.1–1.0 ng/l LOD = 0.1–1.2 ng/l LOD = 0.1–1.2 ng/l LOD = 1.0–15.4 ng/l
[19]
AT, LOV PRA ROS, SV IS = theophylline
Pharmaceutical formulation metabolism study in vitro extraction: ACN:buffer
Interstil ODS 3V (4.6 · 250 mm, 5 lm)
0.01 M AmAc pH 5.0 ACN MeOH
UV 237 nm
–
40
LOQ = 0.1 lg/ml
[20]
AT impurities = DSAT, DFAT, no IS
Bulk drug tablets extraction MeOH
C18 Luna (4.6 · 250 mm, 5 lm)
Gradient elution ACN-ammonium acetate buffer pH 4.0 – THF
UV 248 nm
–
32
LOQ = 0.13 lg/ml LOD = 0.013 lg/ml
[36]
AT, amolodipine + impurities no IS
Tablets MeOH extraction
Perfectsil Target ODS-3 (4.6 · 250 mm, 5 lm)
ACN 0.025 M NaH2HPO4 buffer pH 4.5 (55:45)
UV 237 nm
–
18
LOD = 0.35 lg/ml LOQ = 2 lg/ml
[37]
AT amlodipine No IS
Drug products MeOH extraction
Lichrosphere 100 C18 (4.6 · 250 mm, 5 lm)
50 mM potassium dihydrogen phosphate buffer pH 3.0 ACN (40:60)
UV 254 nm
–
8
LOD = 0.4 lg /ml
[38]
AT IS = DCF
Human serum LLE
Shim-pack CLC-ODS (4.6 · 150 mm, 5 lm)
0.05 M sodium phosphate buffer, pH 4.0 by phosphoric acid MeOH (33:67)
UV 247 nm
–
4
LOQ 4 ng/ml LOD = 1 ng/ml
[39]
AT IS = ibuprofen
Bulk drug tablets human plasma MeOH extraction
RP Supelcosil C18 (4.6 · 150 mm, 5 lm)
ACN:MeOH:water (45:45:10)
UV 240 nm
–
3
LOD = 0.008 lg /ml LOQ = 0. 18 lg/ml
[40]
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Table 2. HPLC analytical methods for the determination of atorvastatin
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Table 2 (continued) Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor ions (notes)
Time [min]
Validation data LOQ /LOD
Ref.
AT novobicin roxytromycin
Aqueous samples SPE
YMC ODS-AQ (1.0 · 100 mm, 3 lm)
Gradient elution A: ACN B: 10 mM AmAc
ESI+ MS-MS SRM
[M + H]+
5.0
ILOQ = 1 pg
[41]
AT, ATA 2-OH-AT/L 4-OH-AT/L IS = deuterium labeled
Human serum LLE
YMC Basic (2.0 · 50 mm, 5 lm)
Gradient elution A: water, MeOH, FAc 88% B: ACN, MeOH, FAc 88% (950 ml:50 ml:43 ll)
ESI+ MS-MS SRM
[M + H]+
3.5
LOQ = 0.5 ng/ml
[42]
AT o-OH-AT, p-OH-AT IS = deuterium labeled
Human, dog and rat plasma LLE
YMC JÕSphere H 80 (C18) (3.0 · 150 mm, 4 lm)
ACN, AcAc 0.1% (70:30)
ESI+ MS-MS SRM
[M + H]+
6
LOQ = 0.250 ng/ml
[43]
AT, ATA p-OH AT/L o-OH AT/L IS = metachalon
Human plasma SPE
Omnisphere C18 (2.0 · 30 mm, 3 lm)
Gradient elution A: ACN, water, FAc 1mM (30:70) B: ACN, water, FAc 1mM (60:40)
ESI+ MS-MS SRM
[M + H]+
21
LOD = 0.06 ng/ml LOD = 0.16 ng/ml o-AT
[18]
AT p-OH AT o-OH AT IS = ROS
Human plasma LLE
Waters Symmetry C18 (4.6 · 100 mm, 5 lm)
0.03% FAc ACN (30:70)
TSI+ MS-MS SRM
[M + H]+
2.5
LOQ = 100 pg/ml
[44]
AT 2-OH-AT IS = clindamycin
Human plasma LLE
Atlantis dC18 (4.0 · 100 mm, 3 lm)
ACN, AcAc (70:30)
ESI+ MS-MS SRM
[M + H]+
4.0
LOD = 0.02 ng/ml LOQ = 0.1 ng/ml
[45]
AT, AT-L p-OH AT/L o-OH AT/L IS = deuterium labeled
Human plasma SPE
4 analytical columns in parallel Genesis C18 (2.2 · 50 mm, 4 lm)
Gradient elution A: 0.1% AcAc in water B: ACN
ESI+ MS-MS SRM
[M + H]+
1.65
–
[46]
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Determined Substances
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Analytical methods for simvastatin and atorvastatin
4 simvastatin atorvastatin
3
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1
0 1992
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2000
2001
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2003
2004
2005
2006
2007
Figure 3. Analytical methods for the determination of simvastatin and atorvastatin – first analytical methods for the determination of simvastatin were published in the early 1990s, and the first for atorvastatin in 1999. The increasing trend for new methods to be developed for both statins can be observed since 2003.
similarity with the molecule of simvastatin (Fig. 4). The difference in the structure is only one methyl group, so such a molecule could also be used as convenient IS in MS detection, since it will have not just the same retention and extraction properties, but also similar ionization in the ion source. Since 2000, there has been a growing number of newly developed methods for the determination of simvastatin, where most of methods employed MS/MS detection (Fig. 3). In the case of simvastatin, there were not only methods for its quantitation also other methods that studied detailed fragmentation [25], chromatographic and mass resolution necessary for analysis of simvastatin and simvastatin acid [26], identification of unknown impurities in simvastatin substance and tablets [27], and the influence of various media (used for simvastatin standard solutions) for the interconversion between simvastatin and simvastatin acid [11]. Quantitative LC-MS methods for simvastatin included, above all, bio-analytical assays [28–34] determining simvastatin in human plasma, one pharmaceutical QC application [27] and one environmental application [19]. They employed MS/MS using a specific SRM (selected reaction monitoring) experiment except for one work, where a single quadrupole was utilized for quantitation enabling only an SIM (selected ion monitoring) experiment for quantitation [30]. Simvastatin in its lactone form was always determined in electrospray positive ion mode (ESI+) or in positive turbo ionspray (TIS+) respectively, while its hydroxy acid form was determined in electrospray negative ion mode
(ESI ), negative turbo ionspray (TIS ) and ESI+ or TIS+. The selection of precursor ion for the determination of simvastatin lactone was not remotely unequivocal – see Tables 1 and 3. Simvastatin forms various adducts influenced by mobile-phase composition, and, unfortunately, such adducts sometimes give higher intensity than [M+H]+, which would be an ideal precursor ion for SRM transition and was utilized in some studies [26,29,32,34]. However, other authors found that simvastatin was not sensitive enough and they observed [M+Na]+, [M+K]+, [M+NH4]+ in the spectra of simvastatin next to the [M+H]+, especially [M+Na]+ and [M+NH4]+, which gave very high intensities [30,31]. They were therefore used as precursor ions for quantitation in some works [28,30]. Favorable experimental conditions could achieve high sensitivity for [M+Na]+ over major molecular ions, as published in earlier LC-MS/MS studies. Nevertheless, the sodium content was found not to be totally controllable during sample analysis. It caused variations in the abundance of the sodium-adduct ion. Sources of sodium (potassium respectively) are believed to originate from the biological matrix or glass containers used during sample preparation and analysis. Sodium acetate could be added to the mobile phase as the way of maintaining better consistency in the abundance of the sodium-adduct ion. Unfortunately, the non-volatile nature of the sodium buffer makes it preferable not to use it during LCMS/MS assays. The factors affecting the relative abundance of [M+Na]+ and [M+H]+ include not only general experimental conditions, such as mobile-phase-buffer http://www.elsevier.com/locate/trac
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O
OH H3C H3C
R=
Lovastatin
R O
O
H
O O CH3
CH3 H3C
H3C
R=
H3C
Simvastatin
O CH3 H3C R=
D313C
[13CD3] Simvastatin
Figure 4. Similarity of simvastatin and lovastatin structures, and stable-isotope labeling of simvastatin.
content and pH, solvents and mass spectrometer parameters, but also the materials of the sample and solution containers. Generally, to reduce sodium content to favor the formation of [M+H]+ ions, containers made of glass and solutions or solvents containing an alcohol, such as methanol, should be avoided throughout sample preparation and analysis [31,35]. There were some attempts to favor the occurrence of [M+NH4]+ by addition of ammonium ions into the mobile phase [31]; however in this extensive study methylammonium adduct [M+CH3NH3]+ was found to be more convenient than other alkylammonium and ammonium adducts tested. It was therefore chosen as a precursor ion in this study and in one other work [33]. Monitored SRM transitions differed among the analytical methods, and the details can be seen in Table 3. The choice of precursor ion is important as well as chromatographic and mass resolution [26]. In the case of simvastatin, where mass unit 18 corresponds the difference between lactone and hydroxy acid form and it is, by coincidence, different by only one mass unit from the 17 mass unit, which corresponds to the difference between [M + H]+ and [M + NH4]+ ions of the lactone or the hydroxy acid form arising in positive ionization mode. The specificity of selected SRM must be verified or chromatographic resolution of the acid and lactone form is required [26]. Most LC-MS quantitative methods determined simvastatin hydroxy acid together with simvastatin, except for one method, where only simvastatin was determined [30]. Simvastatin and its hydroxy acid were separated on C18 analytical column in all cases. Acetonitrile was usually present in mobile phase at more than 70%. Volatile additives including ammonium acetate, formic 362
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acid and methyl-ammonium acetate were added in order to enhance ionization and to get better sensitivity of the method. Ammonium acetate buffer often had pH of 4.5 in order to minimize the interconversion between simvastatin and simvastatin hydroxy acid, because it is much stronger than in the case of atorvastatin. Analytical runs were very short, ranging from 2.5 min up to 5 min. Isocratic elution was applied in most cases. The best ISs for precise and accurate quantitation in MS are stable-isotope-labeled standards. In the case of simvastatin labeling usually occurs at one of the methyl group, [13CD3]-simvastatin is obtained [31,32], see Fig. 4. Four works employed deuterium-labeled ISs [31– 34]. All other works utilized lovastatin and lovastatin acid ISs, as its high stated above, because of its high similarity with the structure of simvastatin – Fig. 4. Method sensitivity expressed as LOQ was typically about 0.05 ng/ml in almost all methods. 4.2. Atorvastatin Analytical methods, even those newly developed, for the determination of atorvastatin and its five possible metabolites, including the lactone form of atorvastatin, p-hydroxyatorvastatin and o-hydroxyatorvastatin and their lactone forms (overview in Table 2), still employ HPLC-UV detection in spite of its lower sensitivity. However, such methods could be successfully applied for the analysis of atorvastatin in drug substances or drug formulations together with impurities, as reported by Ertuk et al. [36], Mohammadi et al. [37] (where amlodipine was also determined in combined tablet formulation), as it was in the work of Chaudhari et al. [38] but without the determination of impurities.
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Table 3. Specific SRM transition for the determination of simvastatin, its various precursor ions, simvastatin hydroxy acid and their stable-isotope labeled forms Compound determined Simvastatin
Molecular weight
Precursor ion +
418.27
[M + H]
[M + Na]+ [M + CH3NH3]+ [M + CH3CN + Na]+ [13CD3]-simvastatin
[M + H]+
422.27
[M + CH3NH3]+ Simvastatin acid
436.27
[M H]
[13CD3]-simvastatin acid
440.27
[M + H]+ [M H]
Two HPLC-UV assays for the determination of atorvastatin in biological materials have been published. Determination of atorvastatin in human serum using diclofenac as the IS was published by Bahrami et al. [39] with an LOQ of 4 ng/ml. Altuntas et al. developed a method for the determination of atorvastatin in human serum, bulk drug and tablets using ibuprofen as the IS with an LOQ of 18 ng/ml [40]. HPLC-UV methods generally utilize the C18 stationary phase. In most cases, a combination of acetonitrile and buffer (ammonium acetate or phosphate) with the aim of keeping pH between 4 and 5. Both isocratic and gradient elution has been utilized. Detection has usually been performed at 237 nm [20,37], 247 nm [36,39] or 254 nm [38]. Analytical run times have been very variable, the shortest about 3–4 min [39,41], the longest about 30 min [36]. None of HPLC-UV methods also determined the active metabolites of atorvastatin or its lactone form.
Specific SRM transition (SIM)
Ref.
419.1 fi 285.1 419.1 fi 199.1 441 (SIM) 450.3 fi 285.1 481.2 fi 440.9
[29,32] [34] [30] [31,33] [28]
423.2 fi 285.1 423.2 fi 199.1 454.3 fi 285.1
[32] [34] [31,33]
435.3 fi 114.0 435.3 fi 319.1 437.3 fi 303 439.2 fi 319.1
[28] [31–34] [29] [31–34]
Fluorescence detection has not been employed in the determination of atorvastatin, probably due to the difficulties with the derivatization step and the repeatability of the procedure. Methods about 10–100 times more sensitive have been developed using MS/MS detection, most of which were for bio-analytical applications, determining atorvastatin in human plasma. One method referred to the determination of atorvastatin together with other drugs (novobiocin and roxytromycin in aqueous samples [41] for the environmental applications). Bio-analytical methods [18,42–46] have, in all cases, employed MS/MS using specific SRM conditions with electrospray ionization in positive-ion mode (ESI+). This may be surprising, taking into account the carboxylic acid group present in the structure of atorvastatin. However, several works confirmed that no higher sensitivity was obtained with electrospray ionization in
Table 4. Specific SRM transition for the determination of atorvastatin, its metabolites and their stable-isotope-labeled forms Compound determined
Molecular weight
Precursor ion +
Specific SRM transition
Ref.
559.2 fi 440.2 564.2 fi 440.2 564.2 fi 445.2 541.2 fi 448.2 546.2 fi 448.2 546.2 fi 453.2
[18,42–46] [42] [43,46] [18,42,46] [42] [46]
Atorvastatin [d5]-atorvastatin
558.25 563.25
[M + H] [M + H]+
Atorvastatin lactone [d5]-atorvastatin lactone
540.25 545.25
[M + H]+ [M + H]+
p-hydroxy atorvastatin [d5]-p-hydroxyatorvastatin p-hydroxyatorvastatin lactone [d5]-p-hydroxyatorvastatin lactone
574.25 579.25 556.25 561.25
[M + H]+ [M + H]+ [M + H]+ [M + H]+
575.2 fi 440.2 580.2 fi 445.2 557.2 fi 448.2 562.2 fi 453.2
[18,42–46] [42,43,46] [18,42,46] [42,46]
o-hydroxyatorvastatin
574.25
[M + H]+
[d5]-o-hydroxyatorvastatin o-hydroxyatorvastatin lactone [d5]-o-hydroxyatorvastatin lactone
579.25 556.25 561.25
[M + H]+ [M + H]+ [M + H]+
575.2 fi 440.2 575.2 fi 466.2 580.2 fi 445.2 557.2 fi 448.2 562.2 fi 453.2
[42–46] [18,44] [42,43,46] [18,42,46] [42,46]
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Table 5. Extraction procedures utilized for sample preparation during simvastatin analysis Matrix
Extraction procedure
Stationary phase LLCE cartridge
SPE eluent/LLE extraction agent
Stabilization/ pre-extraction treatment
Interconversion data
Validation data: recovery
Ref.
SV, SVA LOV, LOVA, PRA
Pu-Ehr tea
SPE
1. Extraction in water at 100C 2. Supelco DSC – C18
90% MeOH
None
Given at various conditions
recSV = 95.0–98.9%
[11]
SV, AT LOV, PRA
Aqueous samples
SPE
1. Filtration 2. Oasis HLB
MeOH
pH 4.5 by 3.0 M H2SO4
1%
recSV = 69–84%
[19]
SV, SVA
Human plasma
SPE
C8
1. MeOH, water 6:4 2. ACN
None
Not significant
recSV = 40% recSVA = 40%
[21]
SV, SVA
Human plasma
LLE
–
ACN:water (60:40) ACN
None
Not given
recSV = 95.2–96.3% recSVA = 92.8–95.1%
[22]
SV
Human plasma
LLE
–
diethylether
10 M HCl
Not given
recSV = 86.9–90.7%
[23]
SV, SVA
Human plasma
SPE
Oasis HLB
ACN 0.1 M AmAc pH 4.5 (75:25)
None
Not given
recSV = 88.8% recSVA = 85.6%
[28]
SV, SVA
Human plasma
on-line SPE
Oasis HLB
ACN 3.0 mM FAc (10:90)
0.1 M sodium acetate pH 4.2
<1%
recSV P 75% recSVA P 38%
[29]
SV
Human plasma
LLE
–
Et-Ac
None
Not given
rec = 101.4%
[30]
SV, SVA
Plasma
LLCE
ChemElut cartridge
MTBE
100 mM AmAc pH 4.5
<0.07%
recSV = 78% recSVA = 87%
[31]
SV, SVA
Human plasma
LLCE
ChemElut cartridge
MTBE
100 mM AmAc pH 4.5
<0.07%
recSV = 66% recSVA = 73%
[32]
SV, SVA
Human plasma
ulv-SPE
Oasis HLB l-elution 96-well SPE plate
ACN:water (95:5)
100 mM AmAc pH 4.5
<0.08%
recSV = 66% recSVA = 73%
[33]
SV, SVA
Human plasma
LLE
96-well LLE plate
MTBE
100 mM AmAc pH 5.0
<0.06% lactonization <0.07% hydrolysis
recSV = 87.1% recSVA = 72.7%
[34]
Trends in Analytical Chemistry, Vol. 27, No. 4, 2008
Substances isolated
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Substances isolated
Matrix
Extraction procedure
Stationary phase
SPE eluent LLE extraction agent
Stabilization /preextraction treatment
Interconversion data
Validation data: recovery
Ref.
AT, SV LOV, PRA
Aqueous samples
SPE
1. Filtration - Soxhlet 2. Oasis HLB
MeOH
pH 4.5 by 3.0 M H2SO4
10%
recAT = 64–86%
[19]
AT
Human serum
LLE
–
Et-Ac
0.1 M phosphate buffer pH 7.0
Not given
recAT = 95%
[39]
AT novobiocin roxytromycin
Aqueous samples
SPE
1. Filtration - Soxhlet 2. HLB
MeOH
pH 4.0 by 3.5 M H2SO4
Not given
recAT = 70.5–72.2%
[41]
AT, ATA 2-OH-AT/L 4-OH-AT/L
Human serum
LLE
–
MTBE
0.1 M sodium acetate buffer pH 5.0
Given at various conditions
rec = 60–100% all analytes
[42]
AT o-OH-AT, p-OH-AT
Human, dog and rat plasma
LLE
–
Diethyl ether
0.1 M NaOH
Given at various conditions
recAT = 100–107%
[43]
AT, ATA p-OH AT/L o-OH AT/L
Human plasma
SPE
Varian C18
ACN 0.1 M AmAc (95:5)
1 M sodium formate pH 3
<4%
rec = 53–78% all analytes
[18]
AT p-OH AT o-OH AT
Human plasma
LLE
–
Diethyl ether dichloromethane (7:3)
10% o-phosphoric acid
Not given
recAT = 54.2%
[44]
AT 2-OH-AT
Human plasma
LLE
–
Diethyl ether dichloromethane (60:40)
0.01 M sodium acetate pH 6.0
Not given
recAT = 89.6–92.5%
[45]
AT, AT-L p-OH AT/L o-OH AT/L
Human plasma
SPE
Isolute C-18
95% MeOH
100 mM AmAc pH 4.6
Not given
Not given
[46]
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Table 6. Extraction procedures utilized for sample preparation during atorvastatin analysis
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negative-ion mode (ESI ), probably because this compound contained two nitrogen groups [42,43]. The precursor ion chosen for quantitation was [M+H]+ in all methods developed (see Table 2). The monitored SRM transition for atorvastatin therefore utilized 559 fi 440 (which was also the most intensive transition in all methods). Compared to HPLC-UV methods, all LC-MS/MS methods determined atorvastatin together with its phydroxy and o-hydroxy metabolites, except [45] where only the o-hydroxy metabolite was determined. All metabolites including hydroxy acid and lactone forms were determined by Jemal et al. [42], Hermann et al. [18] and Van Pelt et al. [46]. Atorvastatin metabolites were all also determined in ESI+ using [M + H]+ as a precursor ion; the specific transition for each metabolite and their stable isotope-labeled forms can be seen in Table 4. Atorvastatin and its metabolites have been separated on the C18 analytical column using acetonitrile – usually about 70% (to shorten retention times of analytes; containing methanol only they would be eluted much later) – as part of the mobile phase, together with acetic or formic acid as additives to enhance ionization. Both isocratic and gradient elution have been utilized. Analytical run times were, in most cases, about 6 min or less, except for one method [18]. The best ISs for precise and accurate quantitation in MS are stable-isotope-labeled standards. In the case of atorvastatin and its metabolites, [d5] labeling usually occurs on the phenyl ring, which does not contain fluorine [42,43]. Only three works employed deuteriumlabeled ISs [42,43,46] because they were very expensive and sometimes they were not easily available. Other works utilized ISs of various structures, including metachalon [18], rosuvastatin [44] or clindamycin [46]. Method sensitivity expressed as LOQ was quite similar for all methods, typically in the range 0.5 ng/ml [42] to 0.1 ng/ml [45]. [Please check this range?]
5. Sample preparation A convenient sample-preparation procedure should isolate the analytes from the complex matrix while removing endogenous interfering substances. This is often the most time-consuming, critical step of analysis. The analytes should be pre-concentrated in order to increase the sensitivity and the selectivity of the method. The procedures for sample preparation of simvastatin and atorvastatin have in most cases included LLE (liquidliquid extraction), and SPE (solid-phase extraction). However, simple extraction into the organic solvent has also been used in pharmaceutical applications [20,24,27,36–38,40] – see Tables 1 and 2. There have also been specific approaches to sample preparation (e.g., 366
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LLCE (liquid-liquid cartridge extraction) [31,32] or online SPE utilizing a column-switching technique). Tables 5 and 6 give overviews of LLE and SPE samplepreparation techniques utilized for the isolation of simvastatin and atorvastatin, focusing on data recovery, interconversion during sample preparation (details given in the work of Herman et al. [18], Zhao et al. [32] and Zhang et al. [34]) and stabilization data. Statins have mostly been isolated using SPE on the Oasis HLB cartridge, employing methanol for elution, or using LLE, where MTBE, ethyl acetate or diethyl ether have generally been applied as extraction agents.
6. Conclusions Analysis of statins is a current problem, because these drugs are widely used for the treatment of hypercholesterolemia. This review has presented HPLC methods for the determination of simvastatin and atorvastatin in various fields of application (pharmacology, clinical medicine and environmental). We divided the methods according to the type of detection. We discussed interconversion between acid and lactone form of statins. LC-MS/MS methods have undoubtedly become the method of choice. In order to get high selectivity and sensitivity, MS/MS techniques employ specific SRM conditions, which are convenient, especially in bioanalytical applications. There is no problem in determining the metabolites, even if they are not also sufficiently separated chromatographically. Chromatographic conditions must be carefully arranged, as must selection of a precursor ion for a specific SRM transition in order not retain the sensitivity and the selectivity of the method.
Acknowledgement The authors gratefully acknowledge the financial support of IGA MZ CR No. 1A/8689-4. References [1] L.Y. Ye, P.S. Firby, M.J. Moore, Ther. Drug. Monit. 22 (2000) 737. [2] Y. Shitara, Y. Sugiyama, Pharmacol. Therapeut. 112 (2006) 71. [3] K.J. Williams, I. Tabas, Atherosclerosis Thrombosis Vascular Biol. 15 (1995) 551. [4] A.A. White, Stats. Med. 14 (1995) 697. [5] D.J. Betteridge, P.M. Dodson, P.N. Durrington, E.A. Hughes, M.F. Laker, D.P. Nicholls, J.A. Rees, C.A. Seymour, G.R. Thompson, A.F. Winder, Postgrad. Med. J. 69 (1993) 359. [6] World Health Organization, World Health Report, Report of the Director-General, WHO, Geneva, Switzerland, 1998. [7] F.M. Sacks, Am. J. Cardiol. 88 (suppl.) (2001) 14N.
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HPLC methods for the determination of simvastatin and atorvastatin Lucie Nova´kova´, Dalibor Sˇatı´nsky´, Petr Solich We review high-performance liquid chromatography (HPLC) methods for the determination of two major statins used in clinical treatment – simvastatin and atorvastatin – in various fields of application, including bio-analytical assays, pharmaceutical assays and environmental applications. Statin molecules are known to be susceptible to interconversion of the lactone and acidic forms, so it is necessary to consider this phenomenon during method development. We highlight liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) methods, as they have become a method of choice in bio-analytical and environmental applications. We compare the methods from the point of view of sensitivity. We discuss selection of the precursor ion for performing selected reaction monitoring (SRM) in MS detection and sample preparation. ª 2008 Elsevier Ltd. All rights reserved. Keywords: Atorvastatin; Bio-analytical method; Environmental analysis; High-performance liquid chromatography; HPLC; Interconversion; Pharmaceutical analysis; Simvastatin; Tandem mass spectrometry Abbreviations: [13CD3], Stable-isotope labeling using 13C and three atoms of deuterium; [d5], Stable isotope labeling using five atoms of deuterium; 2-OH-AT, 2-hydroxyatorvastatin; AcAc, Acetic acid; ACN, Acetonitrile, AmAc, Ammonium acetate; AT, Atorvastatin; AT-L, Atorvastatin lactone; DFAT, Desfluoro-atorvastatin; DSAT, Diastereomer-atorvastatin; ESI, Electrospray ionization; Et-Ac, Ethylacetate; FAc, Formic acid; FD, Fluorescence detection; GC, Gas chromatography; HLB, Hydrophilic lipophilic balance; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HPLC, High-performance liquid chromatography; IS, Internal standard; LDL, Low-density lipoprotein; LLCE, Liquid-liquid cartridge extraction; LLE, Liquid-liquid extraction; LOD, Limit of detection; LOQ, Limit of quantitation; LOV, Lovastatin; LOVA, Lovastatin acid; M, mol/l; MeOH, Methanol; Me- SV, Methyl-simvastatin; MEV, Mevastatin; MP, Mobile phase; MS, Mass spectrometry; MS/MS, Tandem MS; MTBE, Methyl-tert-butyl-ether; ODS, Octadecylsilica, C18; o-OH-AT, o-hydroxyatorvastatin; p-OH-AT, p-hydroxyatorvastatin; PRA, Pravastatin; QC, Quality control; ROS, Rosuvastatin; SDS, Sodium dodecyl-sulphate; SIM, Selected ion monitoring; SPE, Solid-phase extraction; SRM, Selective reaction monitoring; SV, Simvastatin; SVA, Simvastatin acid; THF, Tetrahydrofuran; TIS, Turbo ionspray; ulv, ultra-low volume; UV, Ultraviolet
1. Introduction Lucie Nova´kova´*, Dalibor Sˇatı´nsky´, Petr Solich Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovske´ho 1203, 500 05, Hradec Kra´love´, Czech Republic
*
Corresponding author. Tel.: +420 495067345; Fax: +420 495067164; E-mail: [email protected]
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Statins include natural (lovastatin), semisynthetic (simvastatin, and pravastatin) and synthetic compounds (fluvastatin, atorvastatin, cerivastatin, rosuvastatin and pitavastatin) and are potent, specific and competitive inhibitors of 3-hydroxy-3methlyglutaryl coenzyme A (HMG-CoA) reductase. They are highly effective in reducing total cholesterol and low-density lipoprotein (LDL) cholesterol levels in the human body. HMG-CoA reductase is the key enzyme that catalyzes the conversion of HMG-CoA to mevalonate, which is an early ratelimiting step in the biosynthetic pathway of cholesterol (Fig. 1). High-plasma LDL cholesterol is a risk factor of cardiovascu-
lar diseases, such as atherosclerosis, which is characterized by deposition of cholesterol on the arterial wall [1–3]. Atherosclerosis of the coronary and peripheral vasculature is the leading cause of death worldwide. Statistics show that 38–42% of deaths are related to cardiovascular diseases in Western and other developed countries [4]. Lowering cholesterol levels can arrest or reverse atherosclerosis in all vascular beds and it can significantly decrease the morbidity and the mortality associated with atherosclerosis. The main methods of treating hyperlipidemia (or hypercholesterolemia) are dietary and lifestyle changes and the administration of hypolipidemic drugs [5]. Statins are commonly used to treat several forms of hypercholesterol-
0165-9936/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2008.01.013
Trends in Analytical Chemistry, Vol. 27, No. 4, 2008
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Figure 1. Biosynthesis of cholesterol. Cholesterol is synthesized from acetyl coenzyme A. The synthesis of mevalonate, mediated by HMG-CoA reductase is the rate-limiting step, which regulates the cholesterol synthesis. It is the step where the statin molecule competitively effects the synthesis of cholesterol.
emia.They have potent cholesterol-lowering effects and they could reduce morbidity and mortality associated with coronary heart disease significantly, as proved by many clinical trials [2,6–8]. However, some statins exhibit a number of adverse effects, such as myopathy or rhabdomyolysis, so it is useful to monitor the levels of statins in biological materials in order to establish an appropriate dosage scheme, which would minimize adverse effects and keep the cholesterol-lowering effect [2]. Simvastatin and atorvastatin are the two most commonly occurring drugs in commercially available pharmaceutical formulations used for the clinical treatment of hypercholesterolemia. Their structures can be seen in Fig. 2.
2. Chemistry and pharmacokinetics The first statin molecule – mevastatin – was discovered by Endo et al. in 1976 as a fungal product extracted from Penicillium citrinum [9]. Lovastatin, simvastatin and pravastatin are also derivatives of fungal products. Simvastatin and pravastatin are nowadays produced semi-synthetically from lovastatin and mevastatin. However, fluvastatin is a completely synthetic statin with a very different structure from statins derived from fungal products. It is a mevalonolactone derivative with a fluorophenyl-substituted indole ring. Other synthetic statins (cerivastatin, atorvastatin, rosuvastatin and pitavastatin) have similar structures with fluorophenyl groups. All of totally synthetic statins have open-ring
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Trends in Analytical Chemistry, Vol. 27, No. 4, 2008 OH O CH3
H3C
O
O
H
H3C
O
CH3
H3C
Simvastatin (lactone form)
HO COOH OH
CH3
F N
CH3 H N O
Atorvastatin (acid form)
Figure 2. Chemical structures of simvastatin and atorvastatin.
acid forms [2,9]. All statins are absorbed rapidly following administration, reaching peak plasma concentration within 4 hours. Statins exist in two forms, lactone and open-ring hydroxy acid [10,11]. In vivo, the hydroxy acid forms are the active drugs that lower plasma cholesterol while the lactone forms are inactive (prodrug). The lactone form of statin can be absorbed from the gastrointestinal tract and transformed into the active drugs in liver and non-hepatic tissues [11]. Depending upon chemical structure, statins have different affinities for HMG-CoA reductase, which determines their pharmacological effects and different pharmacokinetic properties (e.g., tissue distribution, metabolic stability, enzymes and transporters involved in their metabolism) [2]. We will provide further information on simvastatin and atorvastatin, the most widely used statins in clinical treatment – Fig. 2. Simvastatin is a prodrug, which is administered as an inactive lactone form. The lactone is absorbed from gastrointestinal tract and hydrolyzed to the active b-hydroxy acid form in the liver. Simvastatin and its hydroxy acid are extensively (95%) bound to plasma proteins. The substance undergoes extensive first-pass metabolism in the liver and is mainly excreted in the bile. About 85% of administered dose has been recovered from the feces as metabolite and about 10–15% from urine, mainly as inactive forms [12,13]. Atorvastatin is administered in the open-ring hydroxy acid form – the active form. It is absorbed from the 354
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gastrointestinal tract and it undergoes extensive firstpass metabolism in the liver. Liver metabolism produces two active hydroxy metabolites – ortho-hydroxyatorvastatin and para-hydroxyatorvastatin – and three inactive metabolites corresponding to the lactone form. More than 98% is bound to plasma proteins. It is excreted mainly in feces via the bile, with a smaller proportion excreted in the urine. About 70% of the total plasma HMG-CoA activity is attributed to active metabolites of atorvastatin, even if their concentrations are very low [12–14]. As apparent from the information above, the levels of statins in biological fluids are very low, probably because only about 5% of dosed statin reaches the systemic circulation. For atorvastatin and simvastatin, the plasma concentrations are typically at ng/ml levels. The active metabolites of atorvastatin are present at plasma concentration corresponding to pg/ml levels [13], the typical concentration range being 0.1–20 ng/ ml.
3. Interconversion of lactone and hydroxy acid forms A number of classes of drugs could potentially undergo interconversion, which may occur during any of the numerous steps of the bio-analytical method: in the biological matrix before collecting aliquots of samples for the analysis; during extraction; during evaporation to dryness or reconstitution; in the solution in the injection vial; and, in the case of MS, in the ion source as well. There are several categories of drugs that could undergo interconversion. HMG-CoA reductase inhibitors are typical examples of such a class, where the interconversion occurs between lactone and open-ring hydroxy acid [10,11]. Another category could be conversion between samples that contain a carboxylic acid and its acylglucuronide [15]. Samples that contain a thiol group and its disulfide may also interconvert to each other [16]. Minimizing the interconversion will depend on the conditions during the bio-analytical procedure, pH being one of the most important. HMG-CoA reductase inhibitor simvastatin is administered in its lactone form, although the active form is open-ring hydroxy acid. Both forms are found in postdose samples. On the other hand, atorvastatin is administered as the open-ring hydroxy acid, but the post-dose samples contain both acid and lactonized forms [10,11,17,18]. For the samples of hydroxy acid chemical structure and the corresponding lactone forms, it is important to maintain pH between 4 and 5 in order to minimize
Trends in Analytical Chemistry, Vol. 27, No. 4, 2008
interconversion. Increasing the pH above 6 facilitates the conversion of lactone to acid (in the ionized form); by contrast, lowering pH facilitates the conversion of acid to lactone or lactone to acid (in the non-ionized form). Most assays utilize pH around 4.5 (see Tables 1 and 2). It is important to optimize the method in order to minimize such interconversion. Unfortunately, even optimal conditions may not totally prevent interconversion. It is thus essential to design the composition of the calibration standards and quality control (QC) samples in terms of the ratio of the concentration of one analyte to that of the other so that the accuracy and precision obtained for QC samples realistically reflect accuracy and precision that will be obtained for the expected samples. Ideally, the composition of the QC samples should be identical to that of real samples. This is difficult to establish at the early stage of assay, where the composition of real samples is not known and will probably change from sample to sample [10]. It is recommended that the example of methoddevelopment design should include QC samples prepared in real biological material containing only the lactone form, only the hydroxy acid form, and both in varying ratios of lactone to hydroxy acid (1:1; 1:10; 10:1; 1:3; and, 3:1) in order to get good method accuracy and precision. Such QC samples should be prepared at various concentration levels inside the range of calibration curve [10]. In this study, the influence of pH (analytical conditions at pH 4.2, 7.3 and 1.8) on interconversion of analytes was described in detail. Under the conditions that showed practically no interconversion, the results for all QC samples gave excellent values for accuracy (up to 8.6% RSD) and precision (up to 8.7% RSD) with no influence of the ratio of both analytes. However, in another experimental design, the results for accuracy and precision were strongly influenced by this ratio, except for 1:1, which was the same ratio as in the calibration standards. Thus, under the conditions that allow interconversion between analytes, a method validated for the quantitation of the two analytes using calibration standards with the ratio of analyte concentration of 1:1 and QC samples with the same 1:1 ratio can be used for accurate measurements of the analytes in real samples only if such samples also contain the two analytes in the ratio 1:1. Therefore, to obtain an accurate indication of the performance of the method for all real samples, it is necessary to use QC samples that cover the entire spectrum of composition of real samples. To summarize, in developing a method for the quantitation of two analytes that can undergo interconversion, the first step is to select the conditions that will eliminate or minimize interconversion. The second step is judicious selection of the composition of the QC samples and the composition of calibration standards, which should cover the spectrum of the composition of real samples [10].
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4. Analytical methods The determination of drugs is a multi-disciplinary task. During the manufacturing process of a drug substance and drug formulation, there is a need for QC analytical methods to include all known and unknown impurities. Bio-analytical methods are necessary for clinical trials, therapeutic drug monitoring and individual dosagescheme adjustment. Recently, there has also been great interest in monitoring pharmaceutical residues in the environment, so environmentally-focused analytical methods are also necessary. Determination of drugs in biological materials is an important step in drug discovery and drug development, as it provides pharmacokinetic information, and is necessary when the treatment-dose schedule and safety margins need to be established. High-performance liquid chromatography (HPLC) together with various types of detection – UV (ultraviolet), FD (fluorescence detection) and MS – has become the method of choice for bio-analytical method development. Gas chromatography (GC) is somewhat suppressed because of the need for a derivatization step prior to analysis in order to obtain volatile derivatives of the drug molecule, which is often not volatile in the case of pharmaceuticals. As expected from the different structures of simvastatin and atorvastatin, analytical methods for their quantitative determination were developed individually. Because of the structural properties, there are not many analytical methods that determine these two compounds together in one analytical run or even in combination with other statin molecules. This is also probably because statins are not used with other statins simultaneously during treatment of hyperlipidemic patients. There are only two works that describe the determination of a number of statin structures together in one analytical run: an environmental application – where simvastatin, atorvastatin, lovastatin and pravastatin (internal standard (IS) = mevastatin) were determined in aqueous samples [19]; and, a pharmaceutical application – where atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin were determined together for QC of pharmaceutical formulations [20]. Theophylline was employed as IS for quantitation. The analytical run took about 40 min. There have been two reviews on analytical methods for the determination of HMG-CoA reductase inhibitors. The first, published in 2003, referred to lovastatin, simvastatin, pravastatin, fluvastatin and atorvastatin [12]. HPLC and GC methods were discussed. Generally, fluorescence and UV detection were applied together with HPLC. Only two methods for the determination of atorvastatin were available at that time. The second http://www.elsevier.com/locate/trac
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Table 1. HPLC analytical methods for the determination of simvastatin Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor ions notes
Time [min]
Validation data LOQ/LOD
Ref.
SV, SVA LOV, LOVA PRA
Pu-Ehr tea SPE
Luna C18 (4.6 · 250 mm, 5 lm)
ACN, water, AcAc (70:30:0.5)
ESI+ MS-MS SRM
Not stated
15
Not given stability and interconversion study
[11]
SV, AT LOV PRA IS = MEV
Aqueous samples SPE
Genesis C18 (2.1 · 50 mm, 3 lm)
Gradient elution A: ACN + 2 mM MeA + 0.1% AcAc B: water + 2 mM MeA + 0.1% AcAc
ESI+ MS-MS SRM
[M + CH3NH3]+ [M + H]+ [M + CH3NH3]+ [M + CH3NH3]+
5.5
LOD = 0.1–1.0 ng/l LOD = 0.1–1.2 ng/l LOD = 0.1–1.2 ng/l LOD = 1.0–15.4 ng/l
[19]
SV AT, LOV PRA, ROS IS = theophylline
Pharmaceutical formulation metabolism study in vitro extraction MeOH
Interstil ODS 3V (4.6 · 250 mm, 5 lm)
0.01 M AmAc pH 5.0 ACN MeOH
UV 237 nm
–
40
LOQ = 0.1 lg/ml
[20]
SV, SVA IS = LOV, LOVA
Human plasma SPE
Capcell Pak C18 (4.6 · 150 mm, 5 lm)
ACN-water (80:20)
FD 360 nm 430 nm
Derivatization by 1-bromoacetylpyrene
35
LOQ = 0.1 ng/ml
[21]
SV, SVA No IS
Human plasma LLE
ODS Hypersil (4.6 · 250 mm, 5 lm)
0.025 M sodium dihydrogenphosphate pH4.5 ACN (35:65)
UV 238 nm
–
10
LOD = 15 ng/ml
[22]
SV IS = LOV
Human plasma LLE
Capcell Pak C18 UG 120U (1.5 · 250 mm, 5 lm)
ACN 20 mM potassium phosphate buffer pH 5.6 (65:35)
–
30
LOQ = 0.5 ng/l
[23]
SV and impurities (SVA, LOV Me-SV, dimmer acetate ester anhydro-SV IS = propyphenazon
Tablets extraction MP
X-Terra (4.6 · 50 mm, 3.5 lm)
Microemulsion: 0.9% diisopropylether 1.7% SDS 7.0% co-surfactant 90% 25 mM di-sodium phosphate pH 7.0
–
25
LOD = 5 ng/ml LOQ = 10 ng/ml (SV and SVA)
[24]
UV 238 nm
UV 238 nm
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Determined substances
Table 1 (continued) Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor ions notes
Time [min]
Validation data LOQ /LOD
Ref.
SV, LOV
Standard solutions
No LC
No LC
ESI+MSMS SRM
[M+H]+its fragmentations using MS
–
Not stated fragmentation study
[25]
SV, SVA
Standard solutions
C18 (3.9 · 50 mm, 5 lm)
ACN, 3.0 mM FAc (70:25)
ESI+ MS-MS SRM
[M + H]+ [M + NH4]+
2.5
Chromatographic and mass resolution study
[26]
SV + impurities No IS
SV substance tablets Extraction ACN/FAc
Zorbax C8 (4.6 · 150 mm, 3.5 lm)
Gradient elution A: 0.085 phosphoric acid B:ACN A: 0.001% FAc in water B: 0.001% FAc in CAN
DAD ESI+ MS-MS
45
Identification of impurities
[27]
SV, SVA IS = LOV, LOVA
Human plasma SPE
Discovery C18 (4.6 · 50 mm, 5 lm)
ACN, MeOH, AmAc 0.1 M (62:10:28)
ESI+/ MS-MS SRM
[M + CH3CN + Na]+ [M H]
4.5
LOQ = 0.03 ng/ml SV LOQ = 0.02 ng/ml SVA
[28]
SV, SVA IS = LOV, LOVA
Human plasma on-line SPE
C18 (3.9 · 50 mm, 5 lm)
ACN, 3.0 mM FAc (70:25)
ESI+ MS-MS SRM
[M + H]+
2.5
LOQ = 0.5 ng/ml SV
[29]
SV IS = LOV
Human plasma LLE
Shim-pack ODS (4.6 · 150 mm, 5 lm)
MeOH–water (9:1)
ESI+ MS SV
[M + Na]+
5
LOD = 0.05 ng/ml
[30]
SV, SVA IS = deuterium labeled
Eects of mobile phase on ionization/ fragmentation plasma LLCE
Kromasil C18 (2.0 · 50 mm, 4 lm)
ACN-buffer 2 mM pH 4.5 (70:30) buffers: AmAc, hydrazine acetate, alkylammonium acetates
ESI+ MS-MS SRM
[M H] SVA various SV: [M + H]+ [M + Na]+ [M + K]+ [M + NH4]+ [M + RNH3]+ R = alkyl
3.5
LOQ = 50 pg/ml
[31]
SV, SVA IS = stable isotope labeled
Human plasma LLCE
Kromasil C18 (2.0 · 50 mm, 5 lm)
ACN AmAc pH 4.5 by FAc (75:25)
TIS + MS-MS SRM
[M + H] SV [M H] + SVA
3.5
LOQ = 50 pg/ml
[32]
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Determined substances
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[34] LOQ = 0.05 ng/ml Kromasil C18 (2.0 · 50 mm, 5 lm) Human plasma LLE SV, SVA IS = stable isotope labeled
ACN 1 mM methyl ammonium acetate pH 4.5 (75:25)
TIS + MS-MS SRM
[M + H] SV [M H] + SVA
3.5
[33] LOQ = 0.05 ng/ml 3.0 [M H] SVA [M + CH3NH3]+ SV TIS+ MS-MS SRM Synergie Max RP (2.0 · 50 mm, 4 lm) Human plasma ulv-SPE
ACN 1 mM methyl ammonium acetate pH 4.5 (80:20)
Precursor ions notes
SV, SVA IS = deuterium labeled
Table 1 (continued)
Stationary phase analytical column Matrix sample preparation Determined substances
Mobile phase
Detection
Time [min]
Ref.
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Validation data LOQ /LOD
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work [13] referred to some MS bio-analytical methods for the determination of statins. Fig. 3 shows the trend in developing new methods for the determination of simvastatin and atorvastatin. Simvastatin has been monitored since the early 1990s, while, due to its later synthesis, atorvastatin has been monitored since 1999. Since 2003, the number of newly developed methods has been increasing for both analytes. 4.1. Simvastatin LC-MS/MS (LC coupled to tandem MS) is nowadays the method of choice for the determination of simvastatin and its hydroxy acid (Table 1). While, in 2003, there was the same number of GC methods (3), HPLC-UV or FD (3) and LC-MS (3) [12], newly developed methods employed MS/MS. Only simvastatin acid (open-acid form, which arises from interconversion during analytical procedure or in the human body as an active form of a prodrug) was monitored together with simvastatin; no other metabolites were described. Whereas the HPLC-FD method [21] using 1-bromoacetylpyrene for the derivatization could be very convenient for bio-analytical purposes with its LOQ of 0.1 ng/ml, the HPLC-UV assay has borderline sensitivity for the quantitation of simvastatin in biological materials because of the poor UV-absorption properties of simvastatin molecule, giving an LOD of 15 ng/ml [22]. A more sensitive HPLC-UV assay for the determination of simvastatin in human plasma was developed later with an LOQ of 0.5 ng/ml [23]. However, lower sensitivity is no problem for quantitation in pharmaceutical preparations. Such a method was developed by Malenovic et al. [24] utilizing a microemulsion mobile phase for quantitation of simvastatin and its six impurities (simvastatin acid, lovastatin, methyl-simvastatin, dimmer of simvastatin, acetate ester of simvastatin and anhydro-simvastatin) in tablets or the one previously cited [20]. HPLC-UV and FD analytical methods utilized C18 analytical columns for the retention/separation of simvastatin/simvastatin acid together with mobile phase containing acetonitrile in combination with phosphate buffer (to maintain pH around 4–5) or with water in an older method [21]. X-terra hybrid stationary phase was employed in one case together with application of a microemulsion mobile phase [24]. Isocratic elution was applied in most analyses. Detection was performed at 238 nm in all cases [22–24]. A fluorescence method utilized 360 nm and 430 nm [21]. Analytical run time was usually very long, starting from 10 min [22] going up to 35 min using FD [21]. Two bio-analytical methods also determined simvastatin acid next to the lactone form [21,22], as did a pharmaceutical QC method, where simvastatin acid was determined among six impurities using IS propyphenazon [24]. Lovastatin was often employed as an IS because of its unique structural
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Determined substances
Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor ions (notes)
Time [min]
Validation data LOQ /LOD
Ref.
AT, SV LOV, PRA IS = MEV
Aqueous samples SPE
Genesis C18 (2.1 · 50 mm, 3 lm)
Gradient elution A: ACN + 2 mM MeA + 0.1% AcAc B: water + 2 mM MeA + 0.1% AcAc
ESI + MS-MS SRM
[M + CH3NH3]+ [M + H]+ [M + CH3NH3]+ [M + CH3NH3]+
5.5
LOD = 0.1–1.0 ng/l LOD = 0.1–1.2 ng/l LOD = 0.1–1.2 ng/l LOD = 1.0–15.4 ng/l
[19]
AT, LOV PRA ROS, SV IS = theophylline
Pharmaceutical formulation metabolism study in vitro extraction: ACN:buffer
Interstil ODS 3V (4.6 · 250 mm, 5 lm)
0.01 M AmAc pH 5.0 ACN MeOH
UV 237 nm
–
40
LOQ = 0.1 lg/ml
[20]
AT impurities = DSAT, DFAT, no IS
Bulk drug tablets extraction MeOH
C18 Luna (4.6 · 250 mm, 5 lm)
Gradient elution ACN-ammonium acetate buffer pH 4.0 – THF
UV 248 nm
–
32
LOQ = 0.13 lg/ml LOD = 0.013 lg/ml
[36]
AT, amolodipine + impurities no IS
Tablets MeOH extraction
Perfectsil Target ODS-3 (4.6 · 250 mm, 5 lm)
ACN 0.025 M NaH2HPO4 buffer pH 4.5 (55:45)
UV 237 nm
–
18
LOD = 0.35 lg/ml LOQ = 2 lg/ml
[37]
AT amlodipine No IS
Drug products MeOH extraction
Lichrosphere 100 C18 (4.6 · 250 mm, 5 lm)
50 mM potassium dihydrogen phosphate buffer pH 3.0 ACN (40:60)
UV 254 nm
–
8
LOD = 0.4 lg /ml
[38]
AT IS = DCF
Human serum LLE
Shim-pack CLC-ODS (4.6 · 150 mm, 5 lm)
0.05 M sodium phosphate buffer, pH 4.0 by phosphoric acid MeOH (33:67)
UV 247 nm
–
4
LOQ 4 ng/ml LOD = 1 ng/ml
[39]
AT IS = ibuprofen
Bulk drug tablets human plasma MeOH extraction
RP Supelcosil C18 (4.6 · 150 mm, 5 lm)
ACN:MeOH:water (45:45:10)
UV 240 nm
–
3
LOD = 0.008 lg /ml LOQ = 0. 18 lg/ml
[40]
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Table 2. HPLC analytical methods for the determination of atorvastatin
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Table 2 (continued) Matrix sample preparation
Stationary phase analytical column
Mobile phase
Detection
Precursor ions (notes)
Time [min]
Validation data LOQ /LOD
Ref.
AT novobicin roxytromycin
Aqueous samples SPE
YMC ODS-AQ (1.0 · 100 mm, 3 lm)
Gradient elution A: ACN B: 10 mM AmAc
ESI+ MS-MS SRM
[M + H]+
5.0
ILOQ = 1 pg
[41]
AT, ATA 2-OH-AT/L 4-OH-AT/L IS = deuterium labeled
Human serum LLE
YMC Basic (2.0 · 50 mm, 5 lm)
Gradient elution A: water, MeOH, FAc 88% B: ACN, MeOH, FAc 88% (950 ml:50 ml:43 ll)
ESI+ MS-MS SRM
[M + H]+
3.5
LOQ = 0.5 ng/ml
[42]
AT o-OH-AT, p-OH-AT IS = deuterium labeled
Human, dog and rat plasma LLE
YMC JÕSphere H 80 (C18) (3.0 · 150 mm, 4 lm)
ACN, AcAc 0.1% (70:30)
ESI+ MS-MS SRM
[M + H]+
6
LOQ = 0.250 ng/ml
[43]
AT, ATA p-OH AT/L o-OH AT/L IS = metachalon
Human plasma SPE
Omnisphere C18 (2.0 · 30 mm, 3 lm)
Gradient elution A: ACN, water, FAc 1mM (30:70) B: ACN, water, FAc 1mM (60:40)
ESI+ MS-MS SRM
[M + H]+
21
LOD = 0.06 ng/ml LOD = 0.16 ng/ml o-AT
[18]
AT p-OH AT o-OH AT IS = ROS
Human plasma LLE
Waters Symmetry C18 (4.6 · 100 mm, 5 lm)
0.03% FAc ACN (30:70)
TSI+ MS-MS SRM
[M + H]+
2.5
LOQ = 100 pg/ml
[44]
AT 2-OH-AT IS = clindamycin
Human plasma LLE
Atlantis dC18 (4.0 · 100 mm, 3 lm)
ACN, AcAc (70:30)
ESI+ MS-MS SRM
[M + H]+
4.0
LOD = 0.02 ng/ml LOQ = 0.1 ng/ml
[45]
AT, AT-L p-OH AT/L o-OH AT/L IS = deuterium labeled
Human plasma SPE
4 analytical columns in parallel Genesis C18 (2.2 · 50 mm, 4 lm)
Gradient elution A: 0.1% AcAc in water B: ACN
ESI+ MS-MS SRM
[M + H]+
1.65
–
[46]
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Determined Substances
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Analytical methods for simvastatin and atorvastatin
4 simvastatin atorvastatin
3
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1
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2006
2007
Figure 3. Analytical methods for the determination of simvastatin and atorvastatin – first analytical methods for the determination of simvastatin were published in the early 1990s, and the first for atorvastatin in 1999. The increasing trend for new methods to be developed for both statins can be observed since 2003.
similarity with the molecule of simvastatin (Fig. 4). The difference in the structure is only one methyl group, so such a molecule could also be used as convenient IS in MS detection, since it will have not just the same retention and extraction properties, but also similar ionization in the ion source. Since 2000, there has been a growing number of newly developed methods for the determination of simvastatin, where most of methods employed MS/MS detection (Fig. 3). In the case of simvastatin, there were not only methods for its quantitation also other methods that studied detailed fragmentation [25], chromatographic and mass resolution necessary for analysis of simvastatin and simvastatin acid [26], identification of unknown impurities in simvastatin substance and tablets [27], and the influence of various media (used for simvastatin standard solutions) for the interconversion between simvastatin and simvastatin acid [11]. Quantitative LC-MS methods for simvastatin included, above all, bio-analytical assays [28–34] determining simvastatin in human plasma, one pharmaceutical QC application [27] and one environmental application [19]. They employed MS/MS using a specific SRM (selected reaction monitoring) experiment except for one work, where a single quadrupole was utilized for quantitation enabling only an SIM (selected ion monitoring) experiment for quantitation [30]. Simvastatin in its lactone form was always determined in electrospray positive ion mode (ESI+) or in positive turbo ionspray (TIS+) respectively, while its hydroxy acid form was determined in electrospray negative ion mode
(ESI ), negative turbo ionspray (TIS ) and ESI+ or TIS+. The selection of precursor ion for the determination of simvastatin lactone was not remotely unequivocal – see Tables 1 and 3. Simvastatin forms various adducts influenced by mobile-phase composition, and, unfortunately, such adducts sometimes give higher intensity than [M+H]+, which would be an ideal precursor ion for SRM transition and was utilized in some studies [26,29,32,34]. However, other authors found that simvastatin was not sensitive enough and they observed [M+Na]+, [M+K]+, [M+NH4]+ in the spectra of simvastatin next to the [M+H]+, especially [M+Na]+ and [M+NH4]+, which gave very high intensities [30,31]. They were therefore used as precursor ions for quantitation in some works [28,30]. Favorable experimental conditions could achieve high sensitivity for [M+Na]+ over major molecular ions, as published in earlier LC-MS/MS studies. Nevertheless, the sodium content was found not to be totally controllable during sample analysis. It caused variations in the abundance of the sodium-adduct ion. Sources of sodium (potassium respectively) are believed to originate from the biological matrix or glass containers used during sample preparation and analysis. Sodium acetate could be added to the mobile phase as the way of maintaining better consistency in the abundance of the sodium-adduct ion. Unfortunately, the non-volatile nature of the sodium buffer makes it preferable not to use it during LCMS/MS assays. The factors affecting the relative abundance of [M+Na]+ and [M+H]+ include not only general experimental conditions, such as mobile-phase-buffer http://www.elsevier.com/locate/trac
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O
OH H3C H3C
R=
Lovastatin
R O
O
H
O O CH3
CH3 H3C
H3C
R=
H3C
Simvastatin
O CH3 H3C R=
D313C
[13CD3] Simvastatin
Figure 4. Similarity of simvastatin and lovastatin structures, and stable-isotope labeling of simvastatin.
content and pH, solvents and mass spectrometer parameters, but also the materials of the sample and solution containers. Generally, to reduce sodium content to favor the formation of [M+H]+ ions, containers made of glass and solutions or solvents containing an alcohol, such as methanol, should be avoided throughout sample preparation and analysis [31,35]. There were some attempts to favor the occurrence of [M+NH4]+ by addition of ammonium ions into the mobile phase [31]; however in this extensive study methylammonium adduct [M+CH3NH3]+ was found to be more convenient than other alkylammonium and ammonium adducts tested. It was therefore chosen as a precursor ion in this study and in one other work [33]. Monitored SRM transitions differed among the analytical methods, and the details can be seen in Table 3. The choice of precursor ion is important as well as chromatographic and mass resolution [26]. In the case of simvastatin, where mass unit 18 corresponds the difference between lactone and hydroxy acid form and it is, by coincidence, different by only one mass unit from the 17 mass unit, which corresponds to the difference between [M + H]+ and [M + NH4]+ ions of the lactone or the hydroxy acid form arising in positive ionization mode. The specificity of selected SRM must be verified or chromatographic resolution of the acid and lactone form is required [26]. Most LC-MS quantitative methods determined simvastatin hydroxy acid together with simvastatin, except for one method, where only simvastatin was determined [30]. Simvastatin and its hydroxy acid were separated on C18 analytical column in all cases. Acetonitrile was usually present in mobile phase at more than 70%. Volatile additives including ammonium acetate, formic 362
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acid and methyl-ammonium acetate were added in order to enhance ionization and to get better sensitivity of the method. Ammonium acetate buffer often had pH of 4.5 in order to minimize the interconversion between simvastatin and simvastatin hydroxy acid, because it is much stronger than in the case of atorvastatin. Analytical runs were very short, ranging from 2.5 min up to 5 min. Isocratic elution was applied in most cases. The best ISs for precise and accurate quantitation in MS are stable-isotope-labeled standards. In the case of simvastatin labeling usually occurs at one of the methyl group, [13CD3]-simvastatin is obtained [31,32], see Fig. 4. Four works employed deuterium-labeled ISs [31– 34]. All other works utilized lovastatin and lovastatin acid ISs, as its high stated above, because of its high similarity with the structure of simvastatin – Fig. 4. Method sensitivity expressed as LOQ was typically about 0.05 ng/ml in almost all methods. 4.2. Atorvastatin Analytical methods, even those newly developed, for the determination of atorvastatin and its five possible metabolites, including the lactone form of atorvastatin, p-hydroxyatorvastatin and o-hydroxyatorvastatin and their lactone forms (overview in Table 2), still employ HPLC-UV detection in spite of its lower sensitivity. However, such methods could be successfully applied for the analysis of atorvastatin in drug substances or drug formulations together with impurities, as reported by Ertuk et al. [36], Mohammadi et al. [37] (where amlodipine was also determined in combined tablet formulation), as it was in the work of Chaudhari et al. [38] but without the determination of impurities.
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Table 3. Specific SRM transition for the determination of simvastatin, its various precursor ions, simvastatin hydroxy acid and their stable-isotope labeled forms Compound determined Simvastatin
Molecular weight
Precursor ion +
418.27
[M + H]
[M + Na]+ [M + CH3NH3]+ [M + CH3CN + Na]+ [13CD3]-simvastatin
[M + H]+
422.27
[M + CH3NH3]+ Simvastatin acid
436.27
[M H]
[13CD3]-simvastatin acid
440.27
[M + H]+ [M H]
Two HPLC-UV assays for the determination of atorvastatin in biological materials have been published. Determination of atorvastatin in human serum using diclofenac as the IS was published by Bahrami et al. [39] with an LOQ of 4 ng/ml. Altuntas et al. developed a method for the determination of atorvastatin in human serum, bulk drug and tablets using ibuprofen as the IS with an LOQ of 18 ng/ml [40]. HPLC-UV methods generally utilize the C18 stationary phase. In most cases, a combination of acetonitrile and buffer (ammonium acetate or phosphate) with the aim of keeping pH between 4 and 5. Both isocratic and gradient elution has been utilized. Detection has usually been performed at 237 nm [20,37], 247 nm [36,39] or 254 nm [38]. Analytical run times have been very variable, the shortest about 3–4 min [39,41], the longest about 30 min [36]. None of HPLC-UV methods also determined the active metabolites of atorvastatin or its lactone form.
Specific SRM transition (SIM)
Ref.
419.1 fi 285.1 419.1 fi 199.1 441 (SIM) 450.3 fi 285.1 481.2 fi 440.9
[29,32] [34] [30] [31,33] [28]
423.2 fi 285.1 423.2 fi 199.1 454.3 fi 285.1
[32] [34] [31,33]
435.3 fi 114.0 435.3 fi 319.1 437.3 fi 303 439.2 fi 319.1
[28] [31–34] [29] [31–34]
Fluorescence detection has not been employed in the determination of atorvastatin, probably due to the difficulties with the derivatization step and the repeatability of the procedure. Methods about 10–100 times more sensitive have been developed using MS/MS detection, most of which were for bio-analytical applications, determining atorvastatin in human plasma. One method referred to the determination of atorvastatin together with other drugs (novobiocin and roxytromycin in aqueous samples [41] for the environmental applications). Bio-analytical methods [18,42–46] have, in all cases, employed MS/MS using specific SRM conditions with electrospray ionization in positive-ion mode (ESI+). This may be surprising, taking into account the carboxylic acid group present in the structure of atorvastatin. However, several works confirmed that no higher sensitivity was obtained with electrospray ionization in
Table 4. Specific SRM transition for the determination of atorvastatin, its metabolites and their stable-isotope-labeled forms Compound determined
Molecular weight
Precursor ion +
Specific SRM transition
Ref.
559.2 fi 440.2 564.2 fi 440.2 564.2 fi 445.2 541.2 fi 448.2 546.2 fi 448.2 546.2 fi 453.2
[18,42–46] [42] [43,46] [18,42,46] [42] [46]
Atorvastatin [d5]-atorvastatin
558.25 563.25
[M + H] [M + H]+
Atorvastatin lactone [d5]-atorvastatin lactone
540.25 545.25
[M + H]+ [M + H]+
p-hydroxy atorvastatin [d5]-p-hydroxyatorvastatin p-hydroxyatorvastatin lactone [d5]-p-hydroxyatorvastatin lactone
574.25 579.25 556.25 561.25
[M + H]+ [M + H]+ [M + H]+ [M + H]+
575.2 fi 440.2 580.2 fi 445.2 557.2 fi 448.2 562.2 fi 453.2
[18,42–46] [42,43,46] [18,42,46] [42,46]
o-hydroxyatorvastatin
574.25
[M + H]+
[d5]-o-hydroxyatorvastatin o-hydroxyatorvastatin lactone [d5]-o-hydroxyatorvastatin lactone
579.25 556.25 561.25
[M + H]+ [M + H]+ [M + H]+
575.2 fi 440.2 575.2 fi 466.2 580.2 fi 445.2 557.2 fi 448.2 562.2 fi 453.2
[42–46] [18,44] [42,43,46] [18,42,46] [42,46]
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Table 5. Extraction procedures utilized for sample preparation during simvastatin analysis Matrix
Extraction procedure
Stationary phase LLCE cartridge
SPE eluent/LLE extraction agent
Stabilization/ pre-extraction treatment
Interconversion data
Validation data: recovery
Ref.
SV, SVA LOV, LOVA, PRA
Pu-Ehr tea
SPE
1. Extraction in water at 100C 2. Supelco DSC – C18
90% MeOH
None
Given at various conditions
recSV = 95.0–98.9%
[11]
SV, AT LOV, PRA
Aqueous samples
SPE
1. Filtration 2. Oasis HLB
MeOH
pH 4.5 by 3.0 M H2SO4
1%
recSV = 69–84%
[19]
SV, SVA
Human plasma
SPE
C8
1. MeOH, water 6:4 2. ACN
None
Not significant
recSV = 40% recSVA = 40%
[21]
SV, SVA
Human plasma
LLE
–
ACN:water (60:40) ACN
None
Not given
recSV = 95.2–96.3% recSVA = 92.8–95.1%
[22]
SV
Human plasma
LLE
–
diethylether
10 M HCl
Not given
recSV = 86.9–90.7%
[23]
SV, SVA
Human plasma
SPE
Oasis HLB
ACN 0.1 M AmAc pH 4.5 (75:25)
None
Not given
recSV = 88.8% recSVA = 85.6%
[28]
SV, SVA
Human plasma
on-line SPE
Oasis HLB
ACN 3.0 mM FAc (10:90)
0.1 M sodium acetate pH 4.2
<1%
recSV P 75% recSVA P 38%
[29]
SV
Human plasma
LLE
–
Et-Ac
None
Not given
rec = 101.4%
[30]
SV, SVA
Plasma
LLCE
ChemElut cartridge
MTBE
100 mM AmAc pH 4.5
<0.07%
recSV = 78% recSVA = 87%
[31]
SV, SVA
Human plasma
LLCE
ChemElut cartridge
MTBE
100 mM AmAc pH 4.5
<0.07%
recSV = 66% recSVA = 73%
[32]
SV, SVA
Human plasma
ulv-SPE
Oasis HLB l-elution 96-well SPE plate
ACN:water (95:5)
100 mM AmAc pH 4.5
<0.08%
recSV = 66% recSVA = 73%
[33]
SV, SVA
Human plasma
LLE
96-well LLE plate
MTBE
100 mM AmAc pH 5.0
<0.06% lactonization <0.07% hydrolysis
recSV = 87.1% recSVA = 72.7%
[34]
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Substances isolated
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Substances isolated
Matrix
Extraction procedure
Stationary phase
SPE eluent LLE extraction agent
Stabilization /preextraction treatment
Interconversion data
Validation data: recovery
Ref.
AT, SV LOV, PRA
Aqueous samples
SPE
1. Filtration - Soxhlet 2. Oasis HLB
MeOH
pH 4.5 by 3.0 M H2SO4
10%
recAT = 64–86%
[19]
AT
Human serum
LLE
–
Et-Ac
0.1 M phosphate buffer pH 7.0
Not given
recAT = 95%
[39]
AT novobiocin roxytromycin
Aqueous samples
SPE
1. Filtration - Soxhlet 2. HLB
MeOH
pH 4.0 by 3.5 M H2SO4
Not given
recAT = 70.5–72.2%
[41]
AT, ATA 2-OH-AT/L 4-OH-AT/L
Human serum
LLE
–
MTBE
0.1 M sodium acetate buffer pH 5.0
Given at various conditions
rec = 60–100% all analytes
[42]
AT o-OH-AT, p-OH-AT
Human, dog and rat plasma
LLE
–
Diethyl ether
0.1 M NaOH
Given at various conditions
recAT = 100–107%
[43]
AT, ATA p-OH AT/L o-OH AT/L
Human plasma
SPE
Varian C18
ACN 0.1 M AmAc (95:5)
1 M sodium formate pH 3
<4%
rec = 53–78% all analytes
[18]
AT p-OH AT o-OH AT
Human plasma
LLE
–
Diethyl ether dichloromethane (7:3)
10% o-phosphoric acid
Not given
recAT = 54.2%
[44]
AT 2-OH-AT
Human plasma
LLE
–
Diethyl ether dichloromethane (60:40)
0.01 M sodium acetate pH 6.0
Not given
recAT = 89.6–92.5%
[45]
AT, AT-L p-OH AT/L o-OH AT/L
Human plasma
SPE
Isolute C-18
95% MeOH
100 mM AmAc pH 4.6
Not given
Not given
[46]
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Table 6. Extraction procedures utilized for sample preparation during atorvastatin analysis
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negative-ion mode (ESI ), probably because this compound contained two nitrogen groups [42,43]. The precursor ion chosen for quantitation was [M+H]+ in all methods developed (see Table 2). The monitored SRM transition for atorvastatin therefore utilized 559 fi 440 (which was also the most intensive transition in all methods). Compared to HPLC-UV methods, all LC-MS/MS methods determined atorvastatin together with its phydroxy and o-hydroxy metabolites, except [45] where only the o-hydroxy metabolite was determined. All metabolites including hydroxy acid and lactone forms were determined by Jemal et al. [42], Hermann et al. [18] and Van Pelt et al. [46]. Atorvastatin metabolites were all also determined in ESI+ using [M + H]+ as a precursor ion; the specific transition for each metabolite and their stable isotope-labeled forms can be seen in Table 4. Atorvastatin and its metabolites have been separated on the C18 analytical column using acetonitrile – usually about 70% (to shorten retention times of analytes; containing methanol only they would be eluted much later) – as part of the mobile phase, together with acetic or formic acid as additives to enhance ionization. Both isocratic and gradient elution have been utilized. Analytical run times were, in most cases, about 6 min or less, except for one method [18]. The best ISs for precise and accurate quantitation in MS are stable-isotope-labeled standards. In the case of atorvastatin and its metabolites, [d5] labeling usually occurs on the phenyl ring, which does not contain fluorine [42,43]. Only three works employed deuteriumlabeled ISs [42,43,46] because they were very expensive and sometimes they were not easily available. Other works utilized ISs of various structures, including metachalon [18], rosuvastatin [44] or clindamycin [46]. Method sensitivity expressed as LOQ was quite similar for all methods, typically in the range 0.5 ng/ml [42] to 0.1 ng/ml [45]. [Please check this range?]
5. Sample preparation A convenient sample-preparation procedure should isolate the analytes from the complex matrix while removing endogenous interfering substances. This is often the most time-consuming, critical step of analysis. The analytes should be pre-concentrated in order to increase the sensitivity and the selectivity of the method. The procedures for sample preparation of simvastatin and atorvastatin have in most cases included LLE (liquidliquid extraction), and SPE (solid-phase extraction). However, simple extraction into the organic solvent has also been used in pharmaceutical applications [20,24,27,36–38,40] – see Tables 1 and 2. There have also been specific approaches to sample preparation (e.g., 366
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LLCE (liquid-liquid cartridge extraction) [31,32] or online SPE utilizing a column-switching technique). Tables 5 and 6 give overviews of LLE and SPE samplepreparation techniques utilized for the isolation of simvastatin and atorvastatin, focusing on data recovery, interconversion during sample preparation (details given in the work of Herman et al. [18], Zhao et al. [32] and Zhang et al. [34]) and stabilization data. Statins have mostly been isolated using SPE on the Oasis HLB cartridge, employing methanol for elution, or using LLE, where MTBE, ethyl acetate or diethyl ether have generally been applied as extraction agents.
6. Conclusions Analysis of statins is a current problem, because these drugs are widely used for the treatment of hypercholesterolemia. This review has presented HPLC methods for the determination of simvastatin and atorvastatin in various fields of application (pharmacology, clinical medicine and environmental). We divided the methods according to the type of detection. We discussed interconversion between acid and lactone form of statins. LC-MS/MS methods have undoubtedly become the method of choice. In order to get high selectivity and sensitivity, MS/MS techniques employ specific SRM conditions, which are convenient, especially in bioanalytical applications. There is no problem in determining the metabolites, even if they are not also sufficiently separated chromatographically. Chromatographic conditions must be carefully arranged, as must selection of a precursor ion for a specific SRM transition in order not retain the sensitivity and the selectivity of the method.
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