HPLC-MS/MS測定城市廢水中的10種藥物與個人護理用品

盧紅選，劉衛(wèi)國,

（1.中國科學院地球環(huán)境研究所 黃土與第四紀地質國家重點實驗室，西安710061；2. 西安交通大學 人居環(huán)境與建筑工程學院，西安 710049）

HPLC-MS/MS測定城市廢水中的10種藥物與個人護理用品

盧紅選1，劉衛(wèi)國1,2

（1.中國科學院地球環(huán)境研究所 黃土與第四紀地質國家重點實驗室，西安710061；2. 西安交通大學 人居環(huán)境與建筑工程學院，西安 710049）

采用固相萃取技術對水樣進行預處理，結合液相色譜-串聯(lián)質譜分析方法（HPLC-MS/ MS），建立了同時檢測城市廢水中包括撲熱息痛、萘普生、磺胺甲惡唑、磺胺二甲嘧啶、三氯生、雙氯芬酸鈉、三氯卡班、鹽酸四環(huán)素、鹽酸土霉素、吉非羅平在內共計10種藥物與個人護理用品（PPCPs）的分析檢測方法。采用中性條件萃取水樣，控制上樣流速為3 — 5 mL · min–1，用甲醇溶液洗脫。純水的平均加標回收率為40.8% — 104.5%，相對標準偏差為5.0% — 25.5%（n = 3）。應用所建立的分析方法，對西安浐河表層水進行了分析。結果表明：該方法可用于城市廢水PPCPs的檢測。10種目標物質中，共檢測到4種，其含量為1.4 — 15.0 ng · L–1。

固相萃?。桓咝б合嗌V-串聯(lián)質譜；藥物與個人護理品；城市廢水

藥物與個人護理品（pharmaceuticals and personal care products，PPCPs）是一類新型的環(huán)境污染物，主要是指各種藥物（如抗生素、類固醇、消炎藥、鎮(zhèn)靜劑、抗癲癇藥、避孕藥、神經興奮劑等）以及個人護理品（如化妝品中的合成香料、顯影劑、遮光劑、驅蚊劑、消毒殺菌劑等）等。研究認為：水環(huán)境中殘留的PPCPs可能會導致人類致癌或過敏性反應，并可能使細菌產生耐藥性及抗生素抗性基因的傳遞和擴散，干擾天然細菌的生態(tài)系統(tǒng)，從而威脅人類健康（Daughton and Ternes，1999）。一系列監(jiān)測研究表明：藥物與個人護理品已經成為環(huán)境中廣泛存在的一類新興污染物，在 河 流（Glen et al，2003；Tixier et al，2003；Yu and Chu，2009；Zhang et al，2011；Wu et al，2014）、 湖 泊（Buser and Theobald，1998；Glen et al，2003；Tixier et al，2003；Blair et al，2013；Fergusonet al，2013；Zhu et al，2013）、海洋（Weigel et al，2002；Del Rosario et al，2014）、地下水（Barnes et al，2008）、城市污水（Carmona et al，2014）、飲用水（Carmona et al，2014）、食品（Wu et al，2012；Baron et al，2014）等樣品中被檢出。由于其本身的特殊性質，它們在環(huán)境中的殘留及潛在風險引起了人們越來越多的憂慮。

傳統(tǒng)的PPCPs的檢測方法有微生物檢測法，分光光度檢測法、氣相色譜、液相色譜、電泳分析法等。自20世紀90年代以來，隨著新型接口技術，如電噴霧離子化（ESI）、大氣壓化學電離（APCI）、大氣壓光電離（APPI）等的成熟，液相色譜-質譜（HPLC-MS）技術在PPCPs檢測研究中得到了廣泛的應用（Tixier et al，2003；Barnes et al，2008；Blair et al，2013；Zhu et al，2013；Carmona et al，2014）。高分辨率或串聯(lián)質譜（MS-MS）可提供目標物質結構的詳細信息，具有高選擇性和高靈敏性，非常適用于環(huán)境樣品中痕量藥物的分析檢測，已成為環(huán)境樣品中PPCPs化合物的分析檢測的一個強有力的工具。據(jù)文獻報道，我國水環(huán)境PPCPs化合物在環(huán)境水樣中檢出的質量濃度通常是ng · L–1到μg · L–1級水平（Yang et al，2011；王丹等，2014），目前報道出來的環(huán)境中檢測到的PPCPs的種類多達160多種，因此，開展快速、靈敏、可靠的PPCPs化合物集成定量檢測方法尤為重要。不同于以往文獻中報道的藥物檢測方法多數(shù)僅適用于同類藥物，本文選取包括抗生素類（磺胺甲惡唑、磺胺二甲嘧啶、鹽酸四環(huán)素、鹽酸土霉素），消炎止痛類（撲熱息痛、萘普生、三氯生、雙氯芬酸鈉、三氯卡班）以及血脂調節(jié)劑（吉非羅平）等在內的共10種藥物與個人護理用品為目標物，基于高效液相色譜串聯(lián)質譜聯(lián)用技術，采用固相萃取技術對樣品進行前處理，建立了城市廢水中PPCPs的分析方法，并以西安一城市廢水為代表，對這一新方法進行了考察。

1 實驗部分

1.1 儀器與試劑

PPCPs標準品（撲熱息痛（ACT）、萘普生（NPX）、吉非羅平（GF）、磺胺甲惡唑（SMX）、磺胺二甲嘧啶（SMT）、三氯生（TCS）、雙氯芬酸鈉（DF）、三氯卡班（TCC）、鹽酸四環(huán)素（TC）、鹽酸土霉素（OTC（購自Dr. Ehrenstorfer公司（德國）；羅紅霉素-d7（RTM-d7）購自于terc公司。甲醇、甲酸為HPLC級，其余藥品或試劑均為分析純。

1.2 樣品前處理

用47 mm的 玻 璃 纖 維 濾 紙（GF/F，Whatman）過濾水樣（400 mL），過濾后的水樣，加入200 μL內標混合液，調節(jié)pH = 7.0。固相萃取時，先用3×5 mL高純水通過SPE小柱（PolyseryHLB，6 mL / 500 mg，CNW）進行活化和平衡，而后將含有內標的過濾液以5 —10 mL · min–1的流速通過SPE小柱。接著將5 mL高純水通過SPE小柱，以清洗小柱，并繼續(xù)抽真空30 min，除去水分。再以1 mL · min–1的流速用甲醇洗脫小柱，洗脫液收集于10 mL具塞玻璃刻度離心管中。最后以高純氮吹掃洗脫液（水浴溫度35℃）至剛好吹干，移入自動進樣樣品瓶并用甲醇定容至0.5 mL，待色譜分析（流程見圖1）。

2 結果與討論

2.1 HPLC-MS/MS條件優(yōu)化

根據(jù)目標化合物的理化性質，選擇ESI為離子源。首先配制1 μg · L–1的10種PPCPs化合物標準液，之后根據(jù)化合物性質，結合文獻報道，選取ESI+或者ESI–模式，以流動注射分析（Flow Injection Analysis，F(xiàn)IA）的方法分別確定各目標抗生素的特征離子對。利用獲取的全部特征離子及離子間的豐度比進行定性分析，以豐度最高的特征離子響應與濃度的關系進行定量分析。10種目標化合物優(yōu)化的質譜參數(shù)見表1。

圖1 水中PPCPs 分析方法流程Fig.1 Flow chart for determining the PPCPs in water

表1 10種目標化合物的詳細信息及HPLC-MS/MS參數(shù)Tab.1 Details of the 10 target compounds and operating parameters of HPLC-MS/MS

2.2 HPLC-MS/MS測試分析

采用高效液相色譜-串聯(lián)質譜（島津HPLCESI-MS/MS 8030）對樣品進行測定。流動相A為甲醇溶液，B為含0.01%甲酸的高純水溶液，流速為0.35 mL · min–1。梯度洗脫順序為：0 — 10 min，20% A和80% B；10 — 35 min，90% A和10% B。柱溫40℃。進樣量10 μL。10種PPCPs化合物的總離子流色譜圖見圖2。

配制1—250 μg · L–16個不同濃度的標準溶液，以目標化合物的濃度為橫坐標，不同濃度目標化合物的峰面積為縱坐標，做線性回歸分析。10種PPCPs化合物的工作曲線、相關系數(shù)和檢出限（3倍信噪比）見表2。結果表明：目標化合物在兩個數(shù)量級濃度范圍內具有很好的線性關系。與文獻報道相比，PPCPs化合物的線性范圍以及檢出限均在合理范圍內，考慮到目前我國水環(huán)境PPCPs化合物在環(huán)境水樣中檢出的質量濃度通常是ng · L–1到μg · L–1級水平，本方法可實際應用于環(huán)境中水樣的檢測。

以高純水為介質，分別做3組平行試驗，利用回收率和空白實驗保證實驗的準確性。取400 mL高純水樣，定量加入500 μL濃度為25 μg· L–1的PPCPs混標溶液和200 μL內標溶液，按照圖1所示流程對樣品進行前處理和儀器分析。結果表明：純水中的平均加標回收率為40.8% — 104.5%，相對標準偏差為5.0% — 25.5%（n=3）。

圖2 10種PPCPs 的總離子流色譜Fig.2 Total ion chromatograms of the 10 PPCPs

表2 10種PPCPs化合物的線性回歸方程、相關系數(shù)及檢出限Tab.2 Regression equations, correlation coeffi cients (r2) and method detection limits of the 10 PPCPs

2.3 實際水樣分析

采集西安市區(qū)浐河表層水，采集時間為2015年10月。按照圖1所示流程進行樣品處理和HPLCMS/MS分析，結果表明：10種PPCPs樣品中，共檢測到4種PPCPs，分別為ACT（1.4 ng · L–1），NPX（1.4 ng· L–1），TCC（4.8 ng · L–1）以及GF（15.0 ng · L–1），其他6種PPCPs未檢測到或者濃度低于方法檢測限。

3 結論

結合文獻報道以及實驗室實測結果，本文建立了利用HPLC-MS/MS分析水樣中10種痕量PPCPs化合物的檢測分析方法，并利用該方法對西安浐河水樣進行了PPCPs化合物的檢測分析。由于環(huán)境中的PPCPs化合物種類繁多，各類化合物的性質差別較大，對樣品前處理以及儀器檢測方法均提出了更多的挑戰(zhàn)，僅用一種方法無法實現(xiàn)所有PPCPs化合物的同時檢測分析。因此，今后將進一步優(yōu)化前處理條件，建立更多類別的PPCPs化合物檢測分析方法，并進一步加強注重遷移轉化規(guī)律及環(huán)境風險方面的研究。

王 丹, 隋 倩, 趙文濤, 等. 2014. 中國地表水環(huán)境中藥物和個人護理品的研究進展[J]. 科學通報, 59 (9): 743 - 751. [Wang D, Sui Q, Zhao W T, et al. 2014. Pharmaceutical and personal care products in the surface water of China: A review [J]. Chinese Science Bulletin, 59(9): 743 - 751.]

Barnes K K, Kolpin D W, Furlong E T, et al. 2008. A national reconnaissance of pharmaceuticals and other organic wastewater contaminants in the United States—ⅠGroundwater [J]. Science of the Total Environment, 402(2 / 3): 192 - 200.

Baron P A, Love D C, Nachman K E. 2014. Pharmaceuticals and personal care products in chicken meat and other food animal products: A market-basket pilot study [J]. Science of the Total Environment, 490: 296 - 300.

Blair B D, Crago J P, Hedman C J, et al. 2013. Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern [J]. Chemosphere, 93: 2116 - 2123.

Buser H R, Theobald M D. 1998. Occurrence of the pharmaceutical drug clofibric acid and the herbicide mecoprop in various Swiss lakes and in the North Sea [J]. Environmental Science and Technology, 32(1): 188 - 192.

Carmona E, Andreu V, Picó Y. 2014. Occurrence of acidic pharmaceuticals and personal care products in Turia River Basin: From waste to drinking water [J]. Science of the Total Environment, 484: 53 - 63.

Daughton C G, Ternes T A. 1999. Pharmaceuticals and personal care products in the environment: agents of subtle change? [J]. Environmental Health Perspectives, 107: 907 - 938.

Del Rosario K L, Mitra S, Humphrey C, et al. 2014. Detection of pharmaceuticals and other personal care products in groundwater beneath and adjacent to onsite wastewater treatment systems in a coastal plain shallow aquifer [J]. Science of the Total Environment, 487: 216 - 223.

Ferguson P J, Bernot M J, Doll J C, et al. 2013. Detection of pharmaceuticals and personal care products (PPCPs) in near-shore habitats of southern Lake Michigan [J]. Science of the Total Environment, 458: 187 - 196.

Glen R B, Helge R, Deborah A G, et al. 2003. Pharmaceuticals and personal care products (PPCPs) in surface and treated waters of Louisiana, USA and Ontario, Canada [J]. Science of the Total Environment, 311(1/2/3): 135 - 139.

Tixier C, Singer H P, Oellers S, et al. 2003. Occurrence and fate of carbamazepine, clofibric acid, diclofenac, ibuprofen, ketoprofenand naproxen in surface waters [J]. Environmental Science and Technology, 37(6): 1061 - 1068.

Weigel S, Kuhlmann J, Huhnerfuss H. 2002. Drugs and personal care products as ubiquitous pollutants: occurrence and distribution of clofibric acid, caffeine and DEET in the North Sea [J]. Science of the Total Environment, 295(1/2/3):131 - 141.

Wu C X, Huang X L, Witter J D, et al. 2014. Occurrence of pharmaceuticals and personal care products and associated environmental risks in the central and lower Yangtze River, China [J]. Ecotoxicology and Environmental Safety, 106: 19 - 26.

Wu X Q, Conkle J L, Gan J. 2012. Multi-residue determination of pharmaceutical and personal care products in vegetables [J]. Journal of Chromatography A, 1254: 78 - 86.

Yang J F, Ying G G, Zhao J L, et al. 2011. Spatial and seasonal distribution of selected antibiotics in surface waters of the Pearl Rivers, China [J]. Journal of Environmental Science and Health Part B: Pesticides Food Contaminants and Agricultural Wastes, 46: 272 - 280.

Yu C P, Chu K H. 2009. Occurrence of pharmaceuticals and personal care products along the West Prong Little Pigeon River in east Tennessee, USA [J]. Chemosphere, 75: 1281 - 1286.

Zhang D D, Lin L F, Luo Z X, et al. 2011. Occurrence of selected antibiotics in Jiulongjiang River in various seasons, South China [J]. Journal of Environmental Monitoring, 13: 1953 - 1960.

Zhu S C, Chen H, Li J N. 2013. Sources, distribution and potential risks of pharmaceuticals and personal care products in Qingshan Lake basin, Eastern China [J]. Ecotoxicology and Environmental Safety, 96: 154 - 159.

Determination of 10 pharmaceuticals and personal care products in waste water by HPLC-MS/MS

LU Hongxuan1, LIU Weiguo1,2
(1. State Key Laboratory of Loess and Quaternary Geology, Institute of Earth Environment, Chinese Academy of Sciences, Xi’an 710061, China; 2. School of Human Settlements and Civil Engineering, Xi’an Jiaotong University, Xi’an 710049, China)

Background, aim, and scope Pharmaceuticals and personal care products (PPCPs) represent a variety of chemical, widely used by consumers on a daily basis which include prescription and non-prescription drugs, cosmetics, cleansers, detergents and fragrance produces. PPCPs are considered potentially hazardous compounds because some are ubiquitous, persistent, and biologically active compounds with recognized endocrine disrupting functions (Daughton and Ternes, 1999). These compounds have been widely detected in various environmental matrices throughout the world including rivers (Glen et al, 2003; Tixier et al, 2003; Yu and Chu, 2009; Zhang et al, 2011; Wu et al, 2014), lakes (Buser and Theobald, 1998; Glen et al, 2003; Tixier et al, 2003; Blair et al, 2013; Ferguson et al, 2013; Zhu et al, 2013), oceans (Weigel et al, 2002; Del Rosario et al, 2014), groundwater (Barnes et al, 2008), waste and drinking water (Carmona et al, 2014) and food (Wu et al, 2012; Baron et al, 2014). Detection methods of PPCP include spectrophotometry, gas chromatography, liquid chromatography and electrophoresis. The recent advances in analytical instrumentation have allowed the unequivocal identifi cation and confi rmation of the presence of any compound at very low levels using LC-MS2.The multiple reaction monitoring (MRM) allows monitoring two transitions between precursor and product ions. It is possible to quantify and confi rm the presence of PPCPs at very low concentration levels. However, due to the absence of offi cial monitoring protocols, there is an increasing demand of analytical methods that allow the determination of those compounds in order to obtain more information regarding their behavior and fate in the environments. Therefore, we proposed here a method for the determination of ten pharmaceuticals and personal care products, including acetaminophen, naproxen, diclofenac, sulfamethoxazole, sulfadimidine, triclosan, triclocarban, tetracycline hydrochloride, oxytetracycline and gemfi brozile, in waste water using high performance liquid chromatography-tandem mass spectrometry. The aim of this work is to develop an effi cient method for determination of various PPCPs in water. Materials and methods Filtered water samples were extracted using solid-phase extraction cartridges (SPEs) extraction. HLB SPEs (Poly-sery HLB, 6 mL / 500 mg, CNW) were conditioned with 3×5 mL of water. Water samples were allowed to pass through the cartridges at a fl ow rate of approximately 5 — 10 mL · min–1. After sample loading, the cartridges were then subsequently dried for 30 min under full vacuum, and subsequently the ten pharmaceutical compounds were eluted with 10 mL of methanol. The residue was dissolved in 0.5 mL of methanol and transferred into vials for analysis. The PPCPs were analyzed using liquid chromatography-mass spectrometry (LC-MS) with a Shimadzu 8030 system equipped with an autosampler and Labsolutions manager software. Results Electrospray ionization (ESI) was used as LC-MS interfaces since it is the most frequently used ionization mode which is a soft ionization technique, suitable for polar and moderately non–polar compounds. Fragmentor, collision energy, and other source parameters were optimized by injecting individual standard solutions into mass spectrometer by fl ow injection analysis (FIA). After that, two different MRM transitions were selected for each compound: one for quantifi cation and one for qualifi cation. These ions were monitored under time scheduled MRM conditions. The analysis was done with electrospray ionization in negative mode (ESI–) for TCC, NPX, TCS, DF, GF and in positive mode (ESI+) for the ACT, SMX, SMT, TC, OTC. The initial mobile phase proportion was 20% A and 80% B where A=methanol and B = formic acid:water 1:9999, held for 10 min. A was then increased linearly to 90% in 25 min. The MRM transitions for different PPCPs are as follows: ACT m/z 151/110; NPX m/z 229/185; SMX m/z 254/156; SMT m/z 279/186; TCS m/z 287/35; DF m/z 294/250; TCC m/z 313/60; TC m/z 445/410; OTC m/z 461/426; GF m/z 249/121. Discussion The linearity of the MSMS detector was tested with matrix extracts containing PPCPs at concentration between 1 μg · L–1and 250 μg · L–1for ACT, NPX and DF, between 1 μg · L–1and 100 μg · L–1for SMX and SMT, between 2.5 μg · L–1and 100 μg · L–1for TCS and TCC, between 2.5 μg · L–1and 250 μg · L–1for TC, OTC and GF. The average recoveries of the target compounds in the spiked pure water samples ranged from 40.8% — 104.5% with the relative standard deviations ranged from 5.0% — 25.5% (n = 3). The waste water sample collected from Chanhe River in Xi’an was investigated as a case study. Among the 10 PPCPs, 4 PPCPs were detected and the concentrations ranged from 1.4 ng · L–1to 15.0 ng · L–1. Conclusions A method using HPLCMS/MS has been developed and validated for determination of 10 PPCPs (TCC, NPX, TCS, DF, GF, ACT, SMX, SMT, TC and OTC) in water. Subsequently, the method was successfully applied to analysis of the investigated chemicals in water samples collected from Chanhe River in Xi’an. Recommendations and perspectives The complexity of the biological matrices and the low concentration levels of these compounds make necessary the use of advanced sample treatment procedures, sample clean-up, to remove potentially interfering matrix components, as well as the concentration of analytes. Increased attention would have to be paid to metabolites generated in the organisms and released into the environment as well as to metabolites generated in the environment itself by biodegradation, photolytic or oxidation reactions.

solid phase extraction; HPLC-MS/MS; pharmaceuticals and personal care products (PPCPs); waste water

LU Hongxuan, E-mail: luhx@ieecas.cn

10.7515/JEE201604010

2016-03-24；錄用日期：2016-06-06

Received Date:2016-03-24;Accepted Date:2016-06-06

國家自然科學基金項目（41572157）

Foundation Item:National Natural Science Foundation of China (41572157)

盧紅選，E-mail: luhx@ieecas.cn