郭杰標, 許楊＊,2

(1.食品科學(xué)與技術(shù)國家重點實驗室,南昌大學(xué),江西南昌 330047;2.南昌大學(xué)中德聯(lián)合研究院,江西南昌 330047)

磷酸二酯酶-5抑制劑檢測方法研究進展

郭杰標1, 許楊＊1,2

(1.食品科學(xué)與技術(shù)國家重點實驗室,南昌大學(xué),江西南昌 330047;2.南昌大學(xué)中德聯(lián)合研究院,江西南昌 330047)

3種獲準上市的磷酸二酯酶-5抑制劑藥物(西地那非、伐地那非和他達那非)在市場上獲得了巨大成功。不法商人把這些藥品違法添加到植物保健品和藥品中謀取利益,對公眾的健康構(gòu)成了威脅。不斷出現(xiàn)的PDE-5抑制劑的結(jié)構(gòu)類似物導(dǎo)致問題變得更加復(fù)雜。目前,在標準品的參照下,TLC、HPLC、LC-MS和MECC等檢測方法可以實現(xiàn)對已知PDE-5藥物的檢測。只有液相/離子阱串聯(lián)質(zhì)譜能夠同時檢測合法的藥物和未知的PDE-5抑制劑類似物。免疫學(xué)檢測方法正成為快速、靈敏、高效檢測PDE-5藥物的新方法。

磷酸二酯酶-5;抑制劑;檢測

植物保健品和藥品由于被認為安全和無副作用,近年來在全世界各地廣受消費者青睞[1],天然壯陽藥品(natural aphrodisiacs)是其中最為活躍的產(chǎn)品類型[2]。目前,天然產(chǎn)品中違法添加磷酸二酯酶-5抑制劑(PDE-5 inhibitors)已經(jīng)成為全球性的問題。

磷酸二酯酶-5(PDE-5)主要存在于陰莖海綿體平滑肌和陰莖血管平滑肌,能夠特異性水解環(huán)磷酸鳥苷(cGMP)。PDE-5藥物通過抑制磷酸二酯酶-5提高cGMP含量,激活cGMP依賴性蛋白激酶(PKG)引起胞內(nèi)鈣濃度降低,從而導(dǎo)致陰莖海綿體及陰莖動脈平滑肌舒張,達到治療陽痿的效果[3]。目前,US-FDA批準上市用于治療陽痿的PDE-5抑制劑包括:枸櫞酸西地那非(Viagra,輝瑞)、鹽酸伐地那非(Levitra,拜爾)和他達那非(Cialis,禮萊)。世界各地在天然產(chǎn)品中檢測到的違法添加PDE-5抑制劑,不僅包括以上3種藥品[4-10],還包括對獲準上市的PDE-5藥品進行了結(jié)構(gòu)衍生化的結(jié)構(gòu)類似物(structural analogues)[6,10,15-25]。作者對在保健品和中成藥中違法添加的PDE-5藥物危害性和檢測方法研究進展情況進行綜述。

1 違法添加PDE-5抑制劑藥物的危害性

在天然壯陽藥品中違法添加PDE-5藥物,對公眾健康構(gòu)成一系列的威脅。PDE-5抑制劑藥物具有一系列副作用,如頭痛、面部潮紅、消化不良、視力模糊和肌肉酸痛[26-27]。美國FDA官方網(wǎng)頁(http://www.fda.gov/medwatch/report.htm.)發(fā)布的藥物不良反應(yīng)信息顯示,服用Viagra可能導(dǎo)致失明(2005年),服用Viagra、Levitra和Cialis會導(dǎo)致聽力突然下降甚至失聰(2007年)。因此,世界各國對PDE-5藥物都嚴格按照處方藥管理,在沒有醫(yī)生指導(dǎo)下服用該類藥品是危險的。其次,PDE-5抑制劑與硝酸酯藥物的相互作用會導(dǎo)致嚴重的低血壓[28-29]。硝酸酯藥物廣泛應(yīng)用于糖尿病、高血壓,高血脂和冠心病的治療,患有這些疾病而又伴隨陽痿癥狀的病人,往往求助于天然壯陽藥品。如果這些病人所服用的天然藥品中含違法添加的PDE-5藥物,將會導(dǎo)致可怕的后果。

對于眾多的PDE-5抑制劑結(jié)構(gòu)類似物,問題顯得更加復(fù)雜。首先,由于這些藥品沒有進行過毒理和安全性實驗,存在很多的未知危害因素[20]。日本厚生省官方網(wǎng)頁(http://www.mhlw.go.jp/english/index.html.)報道了一例服用西地那非類似物(hydroxyl-homosildenafil)導(dǎo)致嚴重肝功能損傷的個案。香港一位28歲男性服用西地那非類似物(acetildenafil)后,導(dǎo)致了嚴重的運動共濟失調(diào)(ataxia)[30]。其次,大多數(shù)PDE-5抑制劑類似物比獲準上市的同類藥物具有更強的藥理活性,如Viagra兩個結(jié)構(gòu)類似物hydroxyl-homosildenafil和homosildenafil對PDE-5的50%抑制濃度(IC50值)分別為3.4 nmol/L和3.8 nmol/L,比Viagra(對PDE-5的IC50值為7.6 nmol/L)藥理活性高一倍多[31]。再者,PDE-5抑制劑類似物由于結(jié)構(gòu)被修飾,導(dǎo)致藥物在體內(nèi)的生物學(xué)分布和藥代動力學(xué)發(fā)生改變,從而增加了危險因素。有報道Levitra的結(jié)構(gòu)類似物piperidenafil代謝穩(wěn)定性和脂溶性增強,使其在體內(nèi)維持長時間的高血藥濃度,并且更加容易通過血腦屏障產(chǎn)生嚴重的視覺障礙[32]。因此,與獲準上市的同類藥品相比,違法添加的PDE-5抑制劑類似物對消費者的健康危害程度更大,必須加強監(jiān)管和篩查。

2 PDE-5藥物篩查方法研究進展

近年來,世界各國的檢測部門和科研機構(gòu)對PDE-5藥物篩查方法進行了大量的研究,主要有以下幾種技術(shù):

2.1 樣品預(yù)處理方法

目前,文獻報道的在復(fù)雜基質(zhì)中萃取富集PDE-5藥物的方法,主要使用甲醇、乙腈、二氯甲烷、二甲基亞砜等有機溶劑進行液-液萃取[7-11]。J. Kim等[11]利用二氯甲烷萃取人體血漿中的西地那非及其代謝產(chǎn)物去甲基西地那非,經(jīng)吹干有機溶劑進行濃縮后進行HPLC檢測,檢出限達到2.0 ng/ mL,偏差系數(shù)小于5.0%,加標回收率大于95%。

P.Dzygiel等[12]利用分子印跡聚合物(molecular imprinted polymers,MIPs)作為固相萃取載體,檢測人體血漿中的西地那非及去甲基西地那非,檢測靈敏度為50 ng/mL,偏差系數(shù)小于10%,加標回收率大于70%。M.H.Guermouche等[13]利用商業(yè)化固相萃取柱(Oasis HLB)富集小鼠血漿中的西地那非及去甲基西地那非,檢測靈敏度達到10 ng/mL,偏差系數(shù)小于5.2%,加標回收率大于93%。

可見,樣品預(yù)處理是違法藥物篩查過程中的重要環(huán)節(jié)。而在需要精確定量的PDE-5藥物體內(nèi)代謝監(jiān)測中,樣品預(yù)處理過程更是確保實驗成敗的關(guān)鍵。

2.2 薄層層析(TLC)技術(shù)

TLC技術(shù)簡單易行,檢測成本低廉,適用大規(guī)模初步鑒定。T.S.Reddy等[14]使用碘化鉍鉀顯色,建立了高效薄層層析(HPTLC)技術(shù),在標準品的參照下,篩查天然藥品中違法添加的PDE-5藥物取得了良好的效果。

2.3 高效液相色譜(HPLC)和液質(zhì)聯(lián)用(LC-MS)技術(shù)

高效液相色譜(HPLC)技術(shù)被廣泛應(yīng)用于PDE-5藥物的篩查[7-13,15-25,32],其最大的優(yōu)勢是可以實現(xiàn)定量檢測,檢測結(jié)果的靈敏度、準確性和精密度能夠達到較高的水平[11,13]。液質(zhì)聯(lián)用(LCMS)[16,19-21]能夠結(jié)合檢品的質(zhì)譜信息對違法添加的PDE-5藥物進行確證檢驗。但是,對于一種結(jié)構(gòu)被修飾過的PDE-5藥物類似物,由于其色譜和質(zhì)譜行為都發(fā)生了很大的改變,僅使用HPLC和LC-MS技術(shù)難以進行有效篩查。US-FDA的J.C.Reepmeyer等[19]報道,在同樣的LC-MS條件下,伐地那非和其類似物piperidenafil的保留時間分別是10. 405 mim和14.050 mim,質(zhì)譜結(jié)果顯示m/z分別為499.3([M+H])和460.3([M+H])。在沒有掌握這種新化合物的檢測資料的情況下,很容易認為檢測結(jié)果來自于基質(zhì)的雜信號,從而產(chǎn)生假陰性的檢測結(jié)果。

2.4 液相/串聯(lián)離子阱質(zhì)譜(LC/ESI-MS/MS)技術(shù)

已有大量文獻報道,利用LC/ESI-MS/MS技術(shù)同時檢測違法添加的Viagra、Levitra和Cialis及其結(jié)構(gòu)類似物[6,10,21,33-34]。該技術(shù)把LC分離得到的檢品用電噴霧電離(ESI)轉(zhuǎn)換為準分子離子,經(jīng)碰撞室(CID)進行解離產(chǎn)生的特征離子碎片在二級質(zhì)譜(MS2)進行檢測。Viagra、Levitra和Cialis及其結(jié)構(gòu)類似物的特征離子碎片的m/z值,綜合分析檢品液相分離結(jié)果以及串聯(lián)質(zhì)譜得到的化合物結(jié)構(gòu)信息,就可以在同一過程中檢測出已知的和未知的PDE-5藥物。US-FDA的S.R.Gratz等[21]利用該技術(shù)檢測了40個在市場中抽查的天然壯陽藥品,篩查出了包括多種PDE-5藥物類似物在內(nèi)的19個陽性結(jié)果。

2.5 通過檢測水解產(chǎn)物分析檢品結(jié)構(gòu)

在有些情況下,即使已經(jīng)檢測出一個檢品的分子式,也難以推斷出其結(jié)構(gòu)式。通過在酸性條件下對檢品進行水解,用LC-MS或GC-MS對水解產(chǎn)物進行分析,掌握該檢品的各水解產(chǎn)物的柱保留時間和m/z值,與已知的PDE-5藥物標準品的水解產(chǎn)物的LC-MS或GC-MS檢測結(jié)果進行比較,可以推測目標檢品的精確結(jié)構(gòu)。J.C.Reepmeyer等[19]報道使用LC-MS分別檢測piperidenafil和伐地那非的水解產(chǎn)物,發(fā)現(xiàn)兩者有4個水解產(chǎn)物完全相同,從而確定piperidenafil是伐地那非的結(jié)構(gòu)類似物。他們還通過比較nor-acetildenafil和西地那非水解產(chǎn)物的LC-MS檢測結(jié)果,鑒別出該檢品是西地那非的結(jié)構(gòu)類似物[20]。香港的Y.H.Lam等[25]發(fā)現(xiàn)的一種未知化合物,其LC-MS檢測柱保留時間與伐地那非的一個水解產(chǎn)物相同,m/z值與該水解產(chǎn)物都是313([M+H]),從而鑒定出該化合物是一種只含母核結(jié)構(gòu)的伐地那非類似物。

2.6 膠束動電毛細管色譜法(MECC)

膠束動電毛細管色譜法(micellar electrokinetic capillary chromatography,MECC)比普通毛細管電泳(capillary electrophoresis,CZE)具有更高的分別率和特異性。Nevado等[35]利用MECC同時檢測西地那非和去甲基西地那非,Flores等[36]利用MECC法同時檢測西地那非、伐地那非和他達那非。兩個方法報道的檢測靈敏度基本與LC相當(dāng),但是3個藥品的遷移時間(migration time)分得很開,顯示出比液相方法高得多的分辨率。變異系數(shù)小于5.3%,加標回收率在98%～107%之間,精確度和準確性良好。

2.7 免疫學(xué)快速檢測方法

儀器檢測方法雖然靈敏、精確和可靠,但需要昂貴設(shè)備且運行費用高,難以在基層工作中大規(guī)模開展。免疫學(xué)檢測方法具有靈敏、特異、快速和廉價的優(yōu)點,已廣泛應(yīng)用于食品和環(huán)境樣品的快速檢測[37-38]。

作者用雜交瘤技術(shù)得到多株針對伐地那非的單克隆抗體,并鑒別出其中一株能識別伐地那非的母核結(jié)構(gòu),從而建立了一種能夠同時檢測伐地那非及其類似物的免疫檢測方法(專利申請?zhí)?00910115192X)。該方法對伐地那非的檢測靈敏度為5.0 ng/mL,在檢品在預(yù)處理過程被稀釋了104倍的情況下,對伐地那非加標量為0.08 mg/g的20種中成藥的檢出率為100%,對20種陰性樣品檢測沒有發(fā)現(xiàn)假陽性。使用該免疫檢測系統(tǒng)對US-FDA贈送的伐地那非類似物piperidenafil標準品進行檢測,發(fā)現(xiàn)方法能夠檢測伐地那非的結(jié)構(gòu)類似物。使用該免疫學(xué)方法對兩個經(jīng)US-FDA證實含piperidenafil的保健品的檢測結(jié)果,與使用LCUV對這兩個樣品的檢測結(jié)果相吻合。該檢測系統(tǒng)還顯示出高度的特異性,與西地那非、他達那非、酚妥拉明、睪酮等壯陽藥品基本沒有交叉反應(yīng)。

3 結(jié) 語

自從1998年第一個PDE-5抑制劑Viagra面世以來,該類藥品在市場上取得了巨大的成功。受利益驅(qū)使,不法商人在天然壯陽產(chǎn)品中違法添加PDE-5抑制劑的行為在全球蔓延。而不斷出現(xiàn)的PDE-5抑制劑類似物,不僅對消費者的健康帶來更大的危害性,而且對檢測工作造成了很大的困難?，F(xiàn)代儀器分析技術(shù)的發(fā)展,為篩查違法添加的PDE-5抑制劑提供了多種靈敏和精確的檢驗手段。但能夠配備LC-MS、GC-MS、MECC和LC/ESIMS/MS等高端設(shè)備的檢驗部門畢竟為少數(shù),在我國這樣的發(fā)展中國家尤其是這樣。靈敏、特異、簡便和價廉的免疫學(xué)快速篩查方法是一個值得關(guān)注的研究方向。

[1]R Yuan,YLin.Traditional Chinese medicine:an approach to scientific proof and clinical validatio[J].Pharmacology& Therapeutics,2000,86(2):191.

[2]Venhuis B J,Blok-Tip L,Kaste D de.Designer drugs in herbal aphrodisiacs[J].Forensic Science International,2008,177 (2):e25.

[3]Fleshner N,Harvey M,Adomat H,et al.Evidence for contamination of herbal erectile dysfunction products with phosphodiesterase-5 inhibitors[J].Urology,2005,174(2):636.

[4]Au A M,Ko R,Boo F O,et al.Screening methods for drugs and heavy metals in Chinese patent medicines[J].Bulletin of Environmental Contamination and Toxicology,2000,65(1):112.

[5]Koh H L,Woo S O.Chinese Proprietary medicine in singapore:regulatory control of toxic heavy metals and undeclared drugs[J].Drug Safety,2000,23(5):351.

[6]Blok-Tip L,Zomer B,Bakker F,et al.Structure elucidation of sildenafil analogues in herbal products[J].Food Additives &Contaminants,2004,21(8):737.

[7]Mikami E,Ohno T,Matsumoto H.Simultaneous identification/determination system for phentolamine and sildenafil as adulterants in soft drinks advertising roborant nutrition[J].Forensic Science International,2002,130(2/3):140.

[8]Lai K C,Liu Y C,Tseng M C,et al.Isolation and identifcation of a sildenafil analogue illegally added in dietary supplements[J].Food&Drug Analalsis,2006,14(1):19.

[9]Zou P,Oh S S Y,Hou P,et al.Simultaneous determination of synthetic phospho-diesterase-5 inhibitors found in a dietary supplement and pre-mixed bulk powders for dietary supplements using high-performance liquid chromatography with diode array detection and liquid chromatography[J].Chromatography A,2006,1104(1-2):113.

[10]Zhu X,Xiao S,Chen B,et al.Simultaneous determination of sildenafil,vardenafil and tadalafil as forbidden components in natural dietary supplements for male sexual potency by high-performance liquid chromatography-electrospray ionization mass spectrometry[J].Chromatography A,2005,1066(1-2):89.

[11]Kim J,Ji H Y,Kim SJ,et al.Simultaneous determination of sildena?l and its active metabolite U K-103,320 in human plasma using liquid chromatography/tandem mass spectrometry[J].Pharmaceutical and Biomedical Analysis,2003,32 (2):317.

[12]Dzygiel P,O’Donnell E,Fraier D,et al.Evaluation of water-compatible molecularly imprinted polymers as solid-phase extraction sorbents for the selective extraction of sildenafil and its desmethyl metabolite from plasma samples[J].Chromatography B,2007,853(1-2):346.

[13]Guermouche M H,Bensalah K.Solid phase extraction and liquid chromatographic determination of sildenafil and N-demethylsidenafil in rat serum with basic mobile phase[J].Pharmaceutical and Biomedical Analysis,2006,40(4):952.

[14]Reddy T S,Reddy A S,Devi P S.Identifcation of a sildenafil in dietary supplements by TLC[J].Planar Chromatography -Modern TLC,2006,52(3):427.

[15]Gratz S R,Gamble B M,Flurer R A.Accurate mass measurement using Fourier transform ion cyclotron resonance mass spectrometry for structure elucidation of designer drug analogs of tadalafil,vardenafil and sildenafil in herbal and pharmaceutical matrices[J].Rapid Communications in Mass Spectrometry,2006;20(15):2317.

[16]Reepmeyer J C,d’Avignon D A.Structure elucidation of thioketone analogues of sildenafil detected as adulterants in herbal aphrodisiacs[J].Pharmaceutical and Biomedical Analysis,2009,49(1):145.

[17]Zou P,Hou P L,Yin S S,et al.Isolation and identification of thiohomosildenafil and thiosildenafil in health supplements [J].Pharmaceutical and Biomedical Analysis,2008,47(2):279.

[18]Mei C L,Yi C L,Jer H L.Identification of a sildenafil analogue adulterated in two herbal food supplements[J].Food&Drug Analysis,2006,14(3):260.

[19]Reepmeyer J C,Woodruff J T.Use of liquid chromatography-mass spectrometry and a hydrolytic technique for the detection and structure elucidation of a novel synthetic vardenafil designer drug added illegally to a“natural”herbal dietary supplement[J].Chromatography A,2006,1125(1):67.

[20]Reepmeyer J C,Woodruff J T.Use of liquid chromatography-mass spectrometry and a chemical cleavage reaction for the structure elucidation of a new sildenafil analogue detected as an adulterant in an herbal dietary supplement[J].Pharmaceutical and Biomedical Analysis,2007,44(4):887.

[21]Gratz S R,Flurer C L,Wolnik K A.Analysis of undeclared synthetic phosphodiesterase-5 inhibitors in dietary supplements and herbal matrices by LC-ESI-MS and LC UV[J].Pharmaceutical and Biomedical Analysis,2004;36(3):525.

[22]Reepmeyer J C,Woodruff J T,Avignon D A.Avignon.Structure elucidation of a novel analogue of sildenafil detected as an adulterant in an herbal dietary supplement[J].Pharmaceutical and Biomedical Analysis,2007,43(5):1615.

[23]Hou P,Zou P,Low M Y,et al.Structural identification of a new acetildenafil analogue from pre-mixed bulk powder intended as a dietary supplement[J].Food Additives&Contaminants,2006;23(6):870.

[24]Zou P,Hou P,Low M Y,et al.Structural elucidation of a tadalafil analogue found as an adulterant of a herbal product [J].Food Additives&Contaminants,2006;23(5):446.

[25]Lam Y H,Poon W T,Lai C K,et al.Identification of a novel vardenafil analogue in herbal product[J].Pharmaceutical and Biomedical Analysis,2008,46(4):804.

[26]Wespes E,Amar E,Hatzichristou D,et al.European association of urology.Guidelines on erectile dysfunction[J].European Urology,2002;41(1):1.

[27]Hellstrom W J,Overstreet J W,Yu A,et al.Tadalafil has no detrimental effect on human spermatogenesis or reproductive hormones[J].Urology,2003;170(3):887.

[28]Kloner R.Erectile Dysfunction and Hypertension[J].International Journal of Impotence Research,2007,19(3):296.

[29]Teixeira C E,Priviero F B,Webb R C.Differential effects of the phosphodiesterase type 5 inhibitors sildenafil,vardenafil,and tadalafil in rat aorta[J].Pharmacology and Experimental Therapeutics,2006;316(2):654.

[30]Poon W T,Lam Y H,Lai C K,et al.Analogues of erectile dysfunction drugs:an under-recognised threat[J].Hong Kong Medical Journal,2007,13(2):359.

[31]Wang J,Jiang Y,Wang Y,et al.Liquid chromatography tandem mass spectrometry assay to determine the pharmacokinetics of aildenafil in human plasma[J].Pharmaceutical and Biomedical Analysis,2007,44(1):231.

[32]Daraghmeh N,Al-Omari M,Badwan A A,et al.Determinaton of sildenafil citrate and related substances in the commercial products and tablet dosage form using HPLC[J].Pharmaceutical and Biomedical Analysis,2001,25(3/4):483.

[33]Tseng M C,Lin J H.Determination of sildenafil citrate adulterated in a dietary supplement capsule by LC/MS/MS[J]. Food&Drug Analysis,2002;10(2):112.

[34]Liang Q L,Qu J,Luo G A,et al.Rapid and reliable determination of illegal adulterant in herbal medicines and dietary supplements by LC/MS/MS[J].Pharmaceutical and Biomedical Analysis,2006,40(2):305.

[35]Nevado J J B,Flores J R,Peňalvo G C,et al.Determination of sildenafil and its metabolite by sample stacking with polarity switching using micellar electrokinetic chromatography[J].Chromatography A,2002,953(1):279.

[36]Flores J R,Nevado J J B,Peňalvo G C,et al.Development of a Micellar electrokinetic capillary chromatography method for the determination of three drugs employed in the erectile dysfunction therapy[J].Chromatography B,2004,811(2): 231.

[37]Acharya D,Dhar T K.A novel broad-specific noncompetitive immunoassay and its application in the determination of total aflatoxins[J].Analytica Chimica Acta,2008,630(1):82.

[38]Jeon M,Paeng I R.Quantitative detection of tetracycline residues in honey by a simple sensitive immunoassay[J].Analytica Chimica Acta,2008,626(2):180.

(責(zé)任編輯:朱明)

Update of Techniques for Screening of PDE-5 Inhibitors as Illegal Adulterant

GUO Jie-biao1, XU Yang＊1,2
(1.State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang 330047,China;2.Sino-German Joint Research Institute,Nanchang University,Nanchang 330047,China)

The success of licensed phosphodiesterase type-5(PDE-5)inhibitor drugs(viz sildenafil,vardenafil and tadalafil)has led to the widespread use as adulterants in herbal dietary supplements or herbal medicines.Which cause a threat to the public health,in this review,the apply progress of TLC,HPLC,LC-MS and MECC in the analysis of known PDE-5 drugs with the standards were summarized.However,only LC-ESI-MS/MS can be competent for the identification of both the licensed PDE-5 drugs and unknown analogues in herbal products simultaneously.It is believed that Immunoassay may be a promising strategy for rapid detecting of undecared PDE-5 drugs in a simple,sensitive and high-throughput way.

PDE-5;inhibitions,determination

R 917

:A

1673-1689(2010)03-0331-05

2009-07-13

國家人事部歸國留學(xué)人員資助計劃項目(2006/164)。

郭杰標(1971-),男,廣東韶關(guān)人,講師,食品科學(xué)與工程博士研究生,主要從事食品安全研究;

Email:sgyuanyuan@163.com

＊通信作者:許楊(1951-),女,安徽樅陽人,教授,博士生導(dǎo)師,主要從事食品安全研究;Email:xuyang1951@163.com