Doi:10.1016/j.chroma.2005.12.03

Journal of Chromatography A, 1119 (2006) 277–284 Study on the conversion of three natural statins from lactone forms to their corresponding hydroxy acid forms and their a Department of Health Diet and Restaurant Management, Chung Shan Medical University, 110, Chien-Kuo N. Rd. Sec. 1,Taichung 402, Taiwan b Institute of Food Science and Technology, National Taiwan University, 1, Roosevelt Rd., Sec. 4, Taipei 106, Taiwan Abstract
Conversions of statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, from lactone forms to their corresponding hydroxy acid form in 0.1N NaOH or 0.05N KOH (prepared with 25, 50, 75, 90% acetonitrile or methanol in water or 100% water) were evaluated.
Results showed that lactone form statins could be transformed almost completely only in alkaline solutions prepared with 25 or 50% acetonitrile.
In all methanolic alkaline solutions, lactone form statins could also be converted entirely, nevertheless, they would be further transformed to themethyl ester of the hydroxy acid form and the transformation increased as methanol rises. When lactone and hydroxy acid forms of statins werein methanol, ethyl acetate, 70% acetonitrile in water (with 0.5% acetic acid or no) for 0–48 h at room temperature or in 100 ◦C water for 0–2 h,lactone form statins were converted to their corresponding hydroxy acids, which were raised as time extends and the highest conversions of themwere about 35% in 100 ◦C water and 70% acetonitrile, slightly transformed for lactone form statins in 70% acetonitrile (with 0.5% acetic acid) after8 h, and the other treatments for all statins showed no significant changes. Interferences would be reduced efficiently when statins were extractedfrom Pu-Erh tea with methanol, ethyl acetate or 100 ◦C water followed by purifying through a C18 solid-phase extraction cartridge. Lovastatin wasthe only statin found in Pu-Erh tea and the highest content of it was found under ethyl acetate extraction. In ethyl acetate and methanol extracts,lovastatin existed merely as lactone form. The lowest content of lovastatin was found in the 100 ◦C water extract of Pu-Erh tea, however, both oflactone and hydroxy acid forms were found to exist in the extract.
2005 Elsevier B.V. All rights reserved.
Keywords: HMG-CoA reductase inhibitor; HPLC; Pu-Erh tea; Statin; Lovastatin; Simvastatin; Pravastatin 1. Introduction
Statins exist in two forms, lactone and hydroxy acid in gen- eral. In vivo, the hydroxy acid forms are the active drugs to 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lower plasma cholesterol while the lactone forms are inac- reductase is the key enzyme that catalyzes the conversion of tive (prodrug). Lactone form of statins can be absorbed from HMG-CoA to mevalonate, which is an early rate-limiting step the gastrointestinal tract and transformed to the active drugs in the cholesterol biosynthetic pathway such as lovas- in liver and non-hepatic tissues Some authors tatin, simvastatin, or pravastatin are potent inhibitors of HMG- also reported that lactone form of statins would be con- CoA reductase, which possess high effectiveness in reducing verted to their corresponding hydroxy acids easily in water total cholesterol and the low-density lipoprotein cholesterol lev- and plasma, moreover, the conversion would be enhanced els in the body. So they are usually used to remedy several forms when they are in alkaline solutions (0.1N NaOH and 0.05N of hypercholesterolemic patients and could reduce morbidity and mortality associated with coronary heart disease (CHD) sig- It is important to determine the contents of statins in plasma and their distribution in tissues for pharmaceutical and clini-cal investigations. The major extraction methods of them areliquid–liquid extraction solid-phase extractionThese extraction methods, however, were suitable ∗ Corresponding author. Tel.: +886 2 23629984; fax: +886 2 33664113.
for only one statin at a time and each statin was extracted under E-mail address: [email protected] (L.S. Hwang).
0021-9673/$ – see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2005.12.031 D.-J. Yang, L.S. Hwang / J. Chromatogr. A 1119 (2006) 277–284 Gas chromatography–mass spectrometry (GC–MS) is one neutralization with 0.1N HCl (prepared with acetonitrile), and of the analytical methods to determine statins Though then kept at −20 ◦C after removal of solvent under nitrogen GC–MS provides rapid identification and quantification, it needs and weighed. The purities of these standards were about 95% a time-consuming derivation step for sample preparation. There- as determined by HPLC coupled with photodiode-array detec- fore, high-performance liquid chromatography–mass spectrom- tor (model 996, Waters Associate, Milford, MA, USA). Their chemical structures are shown in Solvents used for the array detector (PDA) adopted extensively for extraction and analysis of statin compounds including methanol, statins analyses, because they could permit rapid identifica- ethyl acetate, acetic acid and hydrochloric acid were obtained tion and quantification and the samples do not need to be from Tedia Co. (Fairfield, OH, USA). HPLC-grade acetoni- derivatized. Ochiai et al. ve derivatized simvastatin with trile was purchased from Lab-Scan (Dublin, Ireland). Deionized 1-bromoacetylpyrene and then determined it by HPLC with flu- water was prepared with Milli-Q water purification system (Mil- orescence detector. Raja et al. ve determined lovastatin lipore Co., Bedford, MA, USA) it was degassed under vacuum and its oxidation products with capillary electrophoresis (CE).
and filtered through a 0.2 ␮m membrane filter (nylon) prior to Endo and Monacolin the first investigators who iso- use in the HPLC analysis. Sodium hydroxide (NaOH) and potas- lated lovastatin (also called mevinolin) from Monascus ruber.
sium hydroxide (KOH) were purchased from Sigma (St. Louis, Albert et al. ve isolated lovastatin from Aspergillus ter- reus. Since then, there are many kinds of statins isolated orsynthesized. Endo et al. Alberts indicated 2.2. The conversions of statins from lactone forms to their that many strains of Monascus as well as a variety of other fila- corresponding hydroxy acid forms under different alkaline mentous fungi including some species of Doratomyces, Eupeni- cillium, Gymnoascus, Hypomyces, Penicillium, Phoma and Tri-choderma could produce lovastatin.
The method was based on that reported by Brown et al. Pu-Erh tea is a post-fermented tea, produced in Yunnan area, and Shen et al. ov-L or Sim-L (0.5 mg) was added to 0.1N which is in the southwestern part of China. Recently, Pu-Erh tea NaOH or 0.05N KOH (prepared with 25, 50, 75 and 90% ace- has gained popularity in Taiwan because of its anti-atherogenic tonitrile or methanol in water or 100% water) and kept at 45 ◦C effect The manufacture of Pu-Erh tea includes both nat- for 1 h, followed by neutralization with 0.5 mL of 0.05N or ural fermentation and prolonged storage under high moisture 0.1N HCl (prepared with acetonitrile or methanol) before HPLC condition at ambient temperature, which is prone to microbial growth, such as Aspergillus. It is therefore expected that Pu-Erhtea contains bioactive metabolites derived from microorganisms.
2.3. Stability of statins in various solutions Hwang et al. that lovastatin was found in Pu-Erhtea, the content of lovastatin was not determined, however.
Each statin standard (0.5 mg) was added to 0.5 mL of There were neither detailed reports on the conversion of methanol, ethyl acetate, or 70% acetonitrile in water (with or statins from lactone forms to their corresponding hydroxy acid without 0.5% acetic acid) for 0, 1, 2, 3, 8, 16, 24 and 48 h at forms, nor the stabilities of statins in various solution. In the room temperature or added to 100 ◦C water for 0, 0.5, 1or 2 h, present study, we investigated three natural statins (lovastatin, simvastatin and pravastatin) and compared their conversion ratesfrom lactone forms to their corresponding hydroxy acids forms 2.4. Solid-phase extraction of statins under different alkaline solutions conditions, and determinedthe stabilities of these three statins in various solutions. We also The method employed was modified from Miao and Met- developed a C18 solid-phase extraction method for the determi- calfe solid-phase extraction cartridges (500 mg) (Supelco Co., St. Louis, MO, USA) were conditioned by passingthrough 6 mL of methanol followed by 6 mL of water. Mixture 2. Experimental
of the five statin standards (each weighed 0.5 mg) was dissolvedin 1 mL of methanol and loaded onto an outlet blocked DSC- C18 cartridge. After evaporation of solvent with nitrogen gas,the outlet of the cartridge was opened and then eluted with 6 mL Pu-Erh tea was purchased from Yunnan, China. It was ground aliquots of water, 5–95% methanol in water (in the increment of into powder of 40 mesh with a RT08 grinder (Rong-Tsong Co., 5% each time) and 100% methanol. Each eluate was collected Taipei, Taiwan) prior to use. Standard compounds of statin, and analyzed by HPLC in order to find the best eluting solvent lovastatin (lactone form) (Lov-L), simvastatin (lactone form, Sim-L) and pravastatin (hydroxy acid form, Pra-H) were pur-chased from Sigma (St. Louis, MO). Lovastatin (hydroxy acid 2.5. Recoveries of statin standards after solid-phase form, Lov-H) and simvastatin (hydroxy acid form, Sim-H) were prepared in our laboratory from Lov-L and Sim-L, respectively.
Lov-L and Sim-L were reacted with 0.1N NaOH (prepared in Mixtures of the five statin standards (each statin weighed 50% aqueous acetonitrile solution) at 45 ◦C for 1 h followed by 20, 100, 500 and 1000 ng), dissolved in methanol, were loaded D.-J. Yang, L.S. Hwang / J. Chromatogr. A 1119 (2006) 277–284 Fig. 1. Chemical structures of statins.
separately onto DSC-C18 solid-phase extraction cartridges as described above. The cartridges were then washed with 6 mLof water followed by 6 mL of 10% methanol in water for the The method employed was similar to that reported by Shen removal of possible impurities, the statins were eluted using et al. The analyses were carried out with a Hitachi L- 6 mL of 90% methanol in water. Each eluate was concentrated 7100 HPLC pump (Hitachi Instruments Inc., Tokyo, Japan) to dryness in a rotary evaporator at 35 ◦C (B¨uchi Co., Flawil, using a isocratic solvent system consisting of acetonitrile, Switzerland) and re-dissolved in 1 mL of the mobile phase deionized water and acetic acid (70/30/0.5, v/v) as the mobile before HPLC analysis. The recovery of each statin was calcu- phase at a flow rate of 1 mL/min. The stationary phase was lated from the analytical result and the original amount of statin Luna C18 column (4.6 mm i.d. × 250 mm, 5 ␮m) (Phenomenex, Torrance, CA, USA) and the sample injection volume was20 ␮L. A Hitachi L-7420 UV–vis detector (set at 237 nm) 2.6. The extraction efficiency of statins from Pu-Erh tea (Hitachi Instruments Inc., Tokyo, Japan) was used to detect The statins in Pu-Erh tea samples were identified by: (1) Adding a mixture of the five statin standards (each weighed addition of statin standards to sample for co-chromatography 20, 100 or 500 ng) to the 10 g of Pu-Erh tea, and then extracted and (2) comparison of retention time, molecular absorption with 100 mL of methanol or ethyl acetate at room temperature spectra, and mass spectra of the unknown peaks with statin for 24 h or refluxed in water (100 mL) at 100 ◦C for 1 h, respec- standards. Absorption spectra were obtained on a photodi- tively. The extracts were filtered and concentrated to dryness in ode array detector (Model 996, Waters Associate, Milford, a rotary evaporator at 35 ◦C. Each extract was dissolved in 1 mL MA, USA) and mass spectra were resolved on a VG plat- of methanol and then pre-treated with DSC-C18 solid-phase form II LC–MS (Micromass Co., Cheshire, UK) (conditions extraction as described above. The eluate was then subjected of LC–MS: electrospray ionization-positive ion, APCI+ mode, to HPLC analysis of statins contents, and the recoveries were cone voltage = 30 eV, source temperature = 120 ◦C, probe tem- perature = 400 ◦C, discharge needle = 3.3 eV).
D.-J. Yang, L.S. Hwang / J. Chromatogr. A 1119 (2006) 277–284 100% water. Hence, we modified to prepare the alkaline solu-tions with 25, 50, 75 and 90% acetonitrile in water. The standard calibration equations of statins, recoveries and indicates that the conversions of Lov-L and Sim-L are similar quantitative analyses were conducted in triplicate and the mean in either 0.1N NaOH or 0.05N KOH, they can be transformed values were determined. Statistical analyses of the data were almost completely only in alkaline solutions prepared with 25 conducted by the analysis of variance and Duncan’s multiple or 50% acetonitrile in water. In the alkaline solutions prepared with 90% acetonitrile, however, Lov-L and Sim-L had only about29–37% and 35–38% conversions, respectively. Higher amounts 3. Results and discussion
of Lov-H and Sim-H were formed (about 86–87% conversionsfor Lov-H and 87–91% conversions for Sim-H) when the alka- 3.1. The conversions of statins from lactone forms to their line solutions were in 75% acetonitrile in 0.1N NaOH. It appears corresponding hydroxy acid forms under different alkaline that alkaline solutions containing lower concentrations of ace- tonitrile in water will facilitate the conversion. Apparently, bothenough water and appropriate solubility are important for the Brown et al. Shen et al. ve prepared hydroxy complete transformation of lactone form statins to their corre- acids of statins from their lactone forms by adding 0.1N NaOH water solution and 0.05N KOH in aqueous methanol, individu- In this investigation, we found that the conversions would ally. In order to confirm the best alkaline condition, we measured be better if lactone form statins were placed in 0.1N NaOH the preparation of hydroxy acids of statins with 0.1N NaOH or or 0.05N KOH solutions prepared with 25 or 50% acetoni- 0.05N KOH (prepared with 25, 50, 75 and 90% methanol in trile in water. Therefore, we recommend that Lov-H and Sim-H water or 100% water) further. The results showed that Lov-L standards to be prepared from Lov-L and Sim-L standards by and Sim-L could be converted to Lov-H and Sim-H entirely in transformation in 0.1N NaOH or 0.05N KOH (prepared with 25 0.1N NaOH and 0.05N KOH (prepared with 25–90% methanol or 50% acetonitrile in water), around 98% conversion can be in water), nevertheless, they would be further transformed to the methyl ester of the hydroxy acid form and the transfor-mation increased as methanol raised There was only 3.2. Stabilities of statins (lactone and hydroxy acid forms) about 5% methyl ester form of lovastatin (Lov-M) generated when Lov-L was in 0.1N NaOH or 0.05N KOH (preparedwith 25% methanol). When the alkaline was prepared in 90% Because the stabilities of statins during extraction or HPLC methanol, 15% Lov-M was formed. Sim-L also showed the sim- analysis would affect the results of quantification, we studied ilar results. Shen et al. Maio and Metcalfe the stabilities of them in various solutions in advance. Each that methanolysis of lactone form statins and esterification of hydroxy acid or lactone form of statin standard (0.5 mg) was dis- hydroxy acid form statins would take place if they were in solved in methanol, ethyl acetate, or 70% acetonitrile in water methanol. In 100% water, however, the conversions of Lov-L (with or without 0.5% acetic acid), respectively for 0–48 h at and Sim-L to Lov-H and Sim-H were only 90–93%. It is spec- room temperature or added to 100 ◦C water for 0–2 h. Lactone ulated that this is due to the insolubility of Lov-L and Sim-L in form statins showed no significant changes in pure methanol and Fig. 2. HPLC chromatograms of lovastatin treated with 0.05N KOH (prepared in solutions containing different ratios of methanol in water) at 45 ◦C for 1 h: (A)90% methanol; (B) 75% methanol; (C) 50% methanol; and (D) 25% methanol. HPLC conditions: column, Luna C-18 (4.6 mm i.d. × 250 mm, 5 ␮m); mobile phase,acetonitrile/H2O/acetic acid = 70/30/0.5 (v/v/v); flow rate, 1 mL/min; detection, 237 nm.
D.-J. Yang, L.S. Hwang / J. Chromatogr. A 1119 (2006) 277–284 Table 1Comparison of the conversion of statins under different alkaline conditions The original content of each statin (lactone form) was 500 ␮g.
a All values are mean ± SD obtained by triplicate analyses, numbers in parentheses are the % conversion.
b Values bearing different letters in the same column are significantly different (p < 0.05).
ethyl acetate at room temperature even after 48 h. The lactone 27.7%, respectively, while Sim-L to Sim-H were 11.1, 27.9 and form statins would be converted to their corresponding hydroxy 29.4%, respectively In 70% acetonitrile at room tem- acids in 70% acetonitrile and 100 ◦C water, and the conversion perature for 1, 24 and 48 h, the conversions were 5.4, 33.5 and increased with time (In 100 ◦C water for 0.5, 1 34.2% for Lov-L to Lov-H and 4.9, 35.9 and 35.3% for Sim-L to and 2 h, the conversions of Lov-L to Lov-H were 8.9, 25.7 and Sim-H, respectively (Hydroxy acid form statins were Table 2The conversions of statins in water at 100 ◦C The original amount of each statin (lactone form) was 500 ␮g.
a All values are mean ± SD obtained by triplicate analyses, numbers in parentheses are the % conversion.
b Values bearing different letters in the same column are significantly different (p < 0.05).
Table 3The conversions of statins in acetonitrile/water (70/30, v/v) at room temperature The original amount of each statin (lactone form) was 500 ␮g.
a All values are mean ± SD obtained by triplicate analyses, numbers in parentheses are the % conversion.
b Values bearing different letters in the same column are significantly different (p < 0.05).
D.-J. Yang, L.S. Hwang / J. Chromatogr. A 1119 (2006) 277–284 Table 4The conversions of statins in acetonitrile/water/acetic acid (70/30/0.5, v/v) at room temperature The original amount of each statin (lactone form) was 500 ␮g.
a All values are mean ± SD obtained by triplicate analyses, numbers in parentheses are the % conversion.
b Values bearing different letters in the same column are significantly different (p < 0.05).
more stable than the lactone forms; they showed no significant 3.3. Evaluation of solid-phase extraction of statins The results were different from those reported by Shen et Due to Pu-Erh tea crude extracts were more adhesive and al. they indicated that methanolysis would take place if contained a lot of impurities, which would interfere with the the lactone form statins were stored in neat methanol at 25 ◦C HPLC analysis of statins. We modified the method of Miao and for 2 days and the methyl ester of statins would be formed.
Metcalfe evaluated the C18 solid-phase extraction for In our study, however, there were no methyl esters of statins the five statin standards. The results showed that the first eluate, occurred in neat methanol at 25 ◦C for 0–48 h regardless of lac- Pra-H was eluted until the C18 cartridge with 15% methanol, tone or hydroxy acid forms of statins. The transformations of Lov-H, Sim-H, Lov-L and Sim-L were then eluted in turn. All statins from lactone forms to their corresponding hydroxy acids of these statins were completely from the cartridge with 90% were found to be retarded when acetic acid (0.5%) was add to methanol. Therefore, the condition of solid-phase extraction of 70% acetonitrile. In this solution, Lov-L and Sim-L could be statins was that samples (extracts) were dissolved in methanol converted to Lov-H and Sim-H until 8 h storing at room tem- and poured them into an outlet blocked C18 cartridge; after dry- perature The percentage conversion was much lower ing the cartridge with nitrogen, the outlet opened cartridge was than in 70% acetonitrile from 16 to 48 h. Shen et al. washed with 6 mL of water and 6 mL of 10% methanol in turn that lactone and hydroxy acid forms of statins could maintain to remove the polar impurities followed by statins elution with their original structures better without affecting lactonization and hydrolysis when they were in 70% acetonitrile with acetic The recoveries of these statins from the above solid-phase acid (0.5%) at 25 ◦C. Our results confirmed their findings. Ace- extraction were about 95–99% when the amounts of statins pass- tonitrile/water/acetic acid (70/30/0.5, v/v) would therefore be ing through the cartridge were from 20 to 1000 ng We, suitable as the mobile phase for HPLC separation of statins.
therefore, felt confident to employ this solid-phase extraction the HPLC chromatogram of the five statin standards. Ye method for the pretreatment of statins containing samples.
et al. ve investigated the conversions of Lov-L to Lov-H inplasma at 37 ◦C, room temperature, −20 ◦C and −70 ◦C. Their 3.4. Statins contents in Pu-Erh tea results showed that the complete transformation (from Lov-Lto Lov-H) would be for 21.5 and 121.5 h at 37 ◦C and room In order to find the best extracting solvent for statins from temperature, respectively, moreover, Lov-L was not altered sig- Pu-Erh tea, 10 g of Pu-Erh tea was spiked with the five statin nificantly at −20 and −70 ◦C for 4 months.
standards (each weighed 20, 100 and 500 ng) and extracted with Fig. 3. HPLC chromatogram of statin standards. HPLC conditions: same as in D.-J. Yang, L.S. Hwang / J. Chromatogr. A 1119 (2006) 277–284 Table 5The recoveries of statins after passing through the C18 solid-phase extraction cartridge a All data are the means of triplicate analyses.
b Values in parentheses are the coefficient of variation (%).
c Values bearing different letters in the same column are significantly different (p < 0.05).
Table 6The recoveries of added statins in Pu-Erh tea after different solvent extraction a All data are the means of triplicate analyses.
b Values in parentheses are the coefficient of variation (%).
c Extraction condition: room temperature, 24 h.
d Extraction condition: 100 ◦C, 1 h.
f Values bearing different letters in the same column are significantly different (p < 0.05).
100 mL of methanol, or ethyl acetate at room temperature for 1 findings. We extracted the statins, which were added to Pu-Erh day or refluxed in 100 mL water at 100 ◦C for 1 hr. The extracts tea by refluxing in water at 100 ◦C for 1 h. This extraction condi- were filtered and dried in a rotary evaporator. Each dry extract tion might cause more vigorous conversion of Lov-L and Sim-L was dissolved in methanol and then poured onto a C18 cartridge for statins solid-phase extraction under the same condition as Pra-H, the high polar compound was not retained well in the column under the HPLC system, and was interfered by polar With methanol or ethyl acetate as extraction solvent, the impurities of Pu-Erh tea extract easily as UV detection. How- recoveries of statins from Pu-Erh tea were about 90–95% ever, it was decided that Pra-H did not existed in Pu-Erh tea after (They were slightly lower than statins alone LC–MS assistant detection; simvastatin (lactone and hydroxy This slight loss of statins in Pu-Erh tea samples might arise from acid forms) also did not be identified. ws that Pu- the statins remained on the tube wall during extraction from Pu- Erh tea contains lovastatin. The highest content of Lov-L was Erh tea, which contained viscous materials. Therefore, lowerrecovery occurred while adding lower amounts of statins to Pu- Erh tea. It is also noticed that the recoveries of lactone form Statins contents in Pu-Erh tea using different solvent extraction statins using methanol and ethyl acetate as extracting solvent were much higher than extracting by refluxing in water. Lac-tone form statins would be transformed to their corresponding hydroxy acids during 100 ◦C water extraction, therefore Lov-L and Sim-L had much lower recoveries, 55–73% and 52–71%, respectively. In the mean time, Lov-H and Sim-H showed the opposite situation, their recoveries were higher (125–137% and a All values are mean ± SD obtained by triplicate analyses.
123–137%, individually) (Miao and Metcalfe ve Extraction condition: room temperature, 24 h.
used the C18 cartridge to extract statins in surface water, and Extraction condition: 100 ◦C, 1 h.
d Values bearing different letters in the same column are significantly different found the recoveries of Lov-L, Sim-L and Pra-H were 90, 82 and 90%, respectively. These results are different from the present D.-J. Yang, L.S. Hwang / J. Chromatogr. A 1119 (2006) 277–284 found under ethyl acetate extraction (139.26 ng/g dw), followed Acknowledgment
by methanol extraction (135.54 ng/g dw), which was not sig-nificantly different from ethyl acetate extraction. The lowest This research was supported by the National Science Council, content of lovastatin (61.54 ng/g dw) was found under 100 ◦C Taiwan, the Republic of China (project no. NSC 92-2811-B-002- water extraction, however, both of Lov-L (21.12 ng/g dw) and Lov-H (40.42 ng/g dw) were found in the extract. Apparently,some of the Lov-L in Pu-Erh tea was transformed into Lov-H References
during 100 ◦C water extraction. Pu-Erh tea is generally boiledin water to obtain the tea infusion for drinking. Therefore, Pu- [1] L.Y. Ye, P.S. Firby, M.J. Moore, Thre. Drug Monit. 22 (2000) 737.
Erh tea infusion may contain both the lactone and hydroxy acid [2] W.H. Frishman, P. Zime tbaum, J. Nadelmann, J. Clin. Pharmacol. 29 forms of lovastatin. Lovastation has been identified in Pu-Erh [3] P.M. Shen, M.S. Shiao, H.R. Chung, K.R. Lee, Y.S. Chao, V.M. Hunt, tea by Hwang et al. n this study, we quantified the amount [4] World Health Organization, World Health Report, Report of the Director- 4. Conclusion
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solution. However, the conversion could be delayed by adding minor amount of acetic acid to the solution. Lactone form statins [10] S.J. Raja, S. Kreft, B. ˇStrukelj, F. Vreˇcer, Croatica Chem. Acta 76 (2003) could be transformed to their corresponding hydoxy acids com- [11] G. Carlucci, P. Mazzeo, L. Biordi, M. Bologna, J. Pharm. Biomed. Anal.
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(prepared with 25–90% methanol in water), lactone form statins [13] Y. Wu, J. Zhao, J. Heinion, W.A. Korfmacher, A.P. Lapiguera, C.C. Lin, could also be converted entirely, nevertheless, they would be fur- [14] K. Otter, C. Mignat, J. Chromatogr. B 708 (1998) 235.
ther transformed to the methyl ester of the hydroxy acid form and [15] X.S. Miao, C.D. Metcalfe, J. Chromatogr. A 998 (2003) 131.
the transformation increased as methanol raised. Solid-phase [16] M. Jemal, Z. Ouyang, M.L. Powell, J. Pharm. Biomed. Anal. 23 (2000) extraction with C18 cartridge is a suitable pretreatment method for Pu-Erh tea samples to reduce the interference from impurities [17] H. Ochiai, N. Uchiyama, K. Imagaki, S. Hata, T. Kamei, J. Chromatogr.
prior to HPLC analysis of statins. The recoveries of added statins [18] A. Endo, K. Monacolin, J. Antibiot. 32 (1979) 852.
from Pu-Erh tea were about 90–95% when methanol or ethyl [19] A.W. Alberts, J. Chen, G. Kuron, V. Hunt, J. Huff, C. Huff, J. Rothrock, acetate was used as extraction solvent. Boiling water extraction, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monaghan, S. Currie, E.
will cause the statins to transform from lactone form to hydoxy Stapley, G. Albers-Schonberg, O. Hensens, J. Hirshfield, K. Hoogsteen, acid form and thus lower the recoveries to were 52–73% for J. Liesch, J. Springer, Proc. Natl. Acad. Sci. U.S.A. 77 (1980) 3957.
lactone form statins and increase the recoveries of hydroxy acid [20] A. Endo, K. Hasumi, A. Yamada, R. Shimoda, T. Hiroshi, J. Antibiot.
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Erh tea, and the content was 139.26 ng/g dw under ethyl acetate [22] L.S. Hwang, L.C. Lin, N.T. Chen, H.C. Liuchang, M.S. Shiao, ACS

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