Document mB0Ygy4ZgpkQxXDpg54ek3nxB

RECYCLED ssoooo } f ";.i --"% ' in otez?vvw Perfluoroetonoie acid in plasma Author: H-W.Hoppe examiner: Leng D raft 22/11/01 Table of contents 1 Basis of th e procedure Of 2 Devices, chemicals and solutions of 2.1 devices 2.2 chemicals 2.3 solutions 2.4 comparison standards 3 Sampling and sample preparation 4 Conditions of work by gos chromatography 5 Analytic regulation 6 Calibration 7 Computation o f th e result of analysis 8 Standardisation and quality assurance 9 Evaluation of th e procedure 9.1 precision 9.2 accuracy 9,3detection limit of 9.4 influences of noise 10 discussion of th e method I I 1 Basis of the procedure Perfluoroctanoic acid is extracted with pH 10 as ion pair in tert-butylm ethylether and derivatised with benzyl bromide. The determination takes place with GCtMS in the ESS or N d mode. Concentrations of interest for industrial medicine and environmental medicine in th e plasma can be determined accurately. 2 Devices, chemicals, solutions 2,1 Devices HP589Q with Split Splitless injection system and autoinjector HP7673 mass-selective detect* e.g, Kodiak 1200 v. Fa. Bear Heating block with drillings for glass tube Heatable evaporation block with blower shaker \ 000056 centrifuge (3000 - 4000 RPM) vacuum centrifuge (e.g. SpeedVac of SAVANT) variable adjustable 100 A and 1 ml pipettes (e.g. of EPPENDORF) 10 tA 100 A glass syringes (e.g. of Hamilton) Autosampler 2 ml vials (e.g. of HEWLETT-PACKARD) 10 ml glass vials with cap 20 ml glass vials with ground glass stoppers 2,2 chemicals tert.Butylm ethylether (TBME) acetone benzyl bromide Aldrich B17905 ! pig pool serum linear Perfluoroctanoic acid I (PFOS) of Aldrich (Cat.Nr. 171468) Perfluornonanoi acid (PFNS) of Aldrich (Cat.Nr. 394459) 2,3 solutions -0,25 M carbonate buffers pH 10 beginning: 4.2 g NaHC03 (Merck 106329) and 14.3 g Na2C03 Decahydrate (Merck 106391) with w ater ad 200 ml solve - 20 % (0.77 M) Tetrabutylammoniumhydroxide (TBA) in water of Merck (KaTNr. 818759) - 0,5 M Tetrabutylammoniumhydroxide (TBA) in water beginning: 65 ml 20 % TBA + 35 ml water mix 2,3 comparison standards 2.3.1 initial solutions Perfluoroctanoic acid (PFQA): Weigh 25 mg PFOA and with acetone add 25 ml exactly. End concentration: l mg/ml. Perfluornonananoic acid (PFNA): Weigh and with acetone add 25 ml exactly. Final concentration 1 mg/ml. 2.3.2 Perfluoroctanoic acid dilution 1 In 10 ml measuring flasks an acetone, add 500 {APFOA initial solution (1 mg/ml). W ith acetone fill to th e mark. Final concentration: 50 pg/ml. 2.3.3 Perfluoroctanoic acid, dilution 2 000057 In 10 ml measuring flasks add, to 100 pi PFOA dilution 2 (50 ug/ml) Fill to th e mark with acetone. Final concentration 0.5 pg/ml. 2.3.4 Perfluornonanoic acid dilution 1 (internal standard) In 10 ml measuring flasks take 10 pi PFNA initial solution (1 mg/ml). With acetonefill to the mark. Final concentration'- 1 pg/mL 2.3.5 calibration standards PFOA solutions o re made by dilution with pig plasma calibration standards, which contain PFOA concentrations between 5000 and 5 pg/l. Pipetting pattern Volume of th e PFOA dilution 1 (50 pg/ml) volume of th e PFOA dilution 2 (0.5 pg/ml) pig plasma concentration in the comparison standard pi pi ml pg/l 50 aA- 0,5 5000 2 5 pi ~ 0.5 2500 5 pi -- 0.5,500 - 1 0 0 pi 0.5,100 -- 50 pL 0.5 5 0 - 1 0 pL 0,510 - 5pL 0.5 5 - - 0.5 0 For questions according to industrial medicine it is sufficient to s e t calibration standards within th e range 100 to 5000 pg/l. For measurements within the environmental medical purposes, standards a re necessary within th e range of 5 to at least 100 pg/l. w 3 Sampling and sample preparation 3,1 Sampling and sample preparation Sampling: Blood withdrawal e g. by means of EDTA - Sarsted Monovette. The plasma production can be accelerated by centrifugation. The blood withdrawal system should have been examined before use fo r zero blanks. Execution: 1. The beginnings fo r samples and calibration standards take place in 10-ml-Srewinde-Roehrchen 2nd standard internal on 0,5 ml plasma 50 A (PFNA Vend. 2) and 2 ml Q.25-AA-Corbonate-Buffer(pH 10) gives and examines (with indicator paper) 3rd 1ml 0,5-M-TBA-Loesong and 3 to pH value each ml TBME in addition-gives; then approx. 60 min intensively mixes (over head shakers, e.g. HEIDOLPH) 4 th following centrifugation 5th 1.7 ml clear TBME excerpt into a Autosampler ampule transfers and 000058 with approx. 35 C under air flow to dry ones re stric ts (is uncritical) 6. the arrears in 100 (A acetone takes up to put 20 fj\ benzyl bromide th e solution to poinf-in-corrodes transfers and zudeckeln. Then approx. 45 min on approx. 75 C heat up. (when investigations according to industrial medicine th e derivatization can be accomplished alternatively in 500 at acetone.) 7. Measurement in th e 61 mode or NCI mode 4 Conditions of work by gas chromatography Detector- KODIAK 1200 (El mode) gas chromatograph: HP 5 8 9 0 I I capillary column: RTX5; -30 m, 0,25 mm I D- x 0,5 jum film Taegergas: Helium 4,6 form- 11 psi tem perature program: 1 min with 70 oC, then with 10 aC/min on 140 oC, with 30 CJtnm on 290 C and 2 min bake. Injector". Split splitless with 250 oC evaporator tubes: 2 mm I.D., task of sample deactivates: 2 0 Splitless time: 1 min tran sfer LINE: 280 oC electron collision ionization: 70 eV E l MS conditions masses fo r PFOA: m/z 504 and 505 (Qualifier) masses fo r PFNA: m/z 554 NCI MSM5 conditions: PFOA: Decomposition of m/z 413 a fte r 369 PFNA: Decomposition from m/z 463 to 419 Cl gas: Methane; Pouring jerk: 6.5 to rr collision gas: Argon; 1.2 mTorr collision energy: ID V 4 Analytic regulation 2pl is injected in each case in the &C, Usually single injections are sufficient. I f the measured values lie outside of th e linearity of th e calibration curve, then th e samples a r e diluted and again regenerated. In each series a quality inspection sample is analyzed. 6 Calibration For th e production of a calibration curve comparison standards are set in pig plasma in accordance with th e pipetting pattern in section 2.3.5 and regenerated as for plasma samples in section 3 described and analyzed according to section 4 by gas chromatography. 7 Computation o f th e results of analysis On th e basis th e calibration curve, the associated concentration in th e blood is determined. ( 000059 8 Standardisation and quality assurance For quality assurance will proceed, as in th e TRgA 410 of th e dangerous material regulation planned. With th e individual analyses, precision and accuracy controls a re analyzed. 9 Evaluation of th e procedure 9.1 precision For th e determination of th e precision doped pig plasma was examined. Measurements in th e E l mode. Spiking n Precision Precision mg/l into the series From day tons day VK (%) VK (%) 10 15 - 8.2.510 4.77 - 0.5 10 3.29 - 0.110 5.00 9.2 accuracy The correctness of th e procedure was examined by regaining experiments with doped individual Human plasma proben. Measurements in the E l mode. I Spiking n Recovery guesses mg/1 Mean (rank) 1010 97 (89,1-107) 2 5 10 98.1 (78-107) 0.510 99 (80-110) 0.0510 93 (78-116) Selectivity comparison. Measurement of PFOA in th e plasma vocationally not more exposed with EI-M5 and NCI MSMS. Person EI-GCMS NCI MSMS pg/J jug/11,9.8 10.8 2 5.4,6.6 3 3 3.5 4 4.3,4.8 5 3.5 5 6 2 3 .1 7 1.5.1 8 5.2 7 9 3 3.710 4.8,5.4 blank 05,0.6 9.3 Detection limits Under th e indicated conditions of work and under taking a signal-to-noise ratio as a basis of 3:1 th e detection limits fo r PFOA amount to 3 pg/l. in th e E l mode. In th e NCI MSMS mode is given the NWG a t present by a blank value a t a value of approx, 0.5 - to 1 pg/l. Those extrapolates detection limit amounts to approx, 0.2 pg/l. 9.4 Influences of noise PFOA of te s t empties values a t a value of approx. 0.5-1 were observed 000060 fjg/l The causes a re not finally clarified yet. The pig plasma used for calibration purposes is unloaded (PFOA < 1 pg/l) and from the blood withdrawal system no PFOA is se t free. Adsorption a t Glass - or or plastic materials one did not observe. 10 Discussion of th e method The available GC-MS method been based on th e procedure of M. Harvhijaervi e t aluminium (1) fo r th e quantification of PFOA by means of extraktive alkylation. From this th e acid anion is extracted a fte r integration with Tetrabutylammoniumhydroxide (TBA) as ion pair from th e plasmq and derivatised afterw ards to th e benzyl ester. Already of M. Hanhijaervi et th e aluminium (1) optimized buffer and i TBA concentrations was invariably taken over. By the change of ethyl I ace ta te against te r t. Butylmethylether (TBME) as extracting (gents th e extraction tem perature of 50 C could on ambient temperature be lowered and th e evaporation of the excerpt to dry ones before the Deriva+isierungssdhritt be substantially accelerated. A fter derivatisation with benzyl bromide in acetone the reaction mixture without fu rth e r cleaning is chromatographed by means of GC-MS on a RTX5 3 0 m x 0.25 mm x 0.5 pm film thickness. GC columns with thinner film, e.g. HP5-MS 30 m x 0.25 mm x 0.25 pm, could be alternatively used. However th e measuring solution had to be diluted of 0,1 to 0,5 ml, since in th e more concentrated solution the benzyl bromide remainders unfavorably affected th e peak form of PFOA with splitless injection (overloading th e column), Detection can tak e place with a MSD in th e EGG or NCI mode (see spectra). I f highest sensitivity is required, NCIGCMS or NCI GCtASMS is recommendable. The dtection limit fo r NCI GCMS is given a t present however by a te s t empties value in Hoehhe of approx. 0.5 pg/l. A parallel measurement with EI-GCMS and NCI GCMSMS a t 10 did not load vocationally show th a t also with EGG-OFFICE-MS environmental medically relevant concentrations can be obtained a t present sensitively and selectively. The determined PFOA of plasma results (2 - 11 ppb, n=!Q) is appropriate for a current HPLC MSMS study (Lit 2) in the lower range of values (< 5 to 35.2 ppb, n = 65). During specific working place loading PFOA plasma concentrations within th e lower ppm range were determined. 000061 The procedure is uncomplicated, very sensitive and durable. The procedure characteristic # data is good as very to designate, t h e procedure works is extended fu rth er to other fluoridated carbonic acids e.g. Perfluorheptane -, Perfuornonane and Prfluordecane acids. Likewise meaning Perfluoroctansulfononic add can be determined however only by means of WPLC-MS (2). Literature 1) M.YIinen, H.Hanhijaervi e t aluminium: Quantitative office determination OF Perfluorooctanic Acid as th e bnzyle ester in plasma and Urine. Arch.Environ.Contam.Toxicol. 14,713-717 (1985) 2) K. J . Hansen e t aluminium: Compound Specific, quantitative Characterization OF Organic Fluorochemicals in Biological Matrices. Environmental Science & Technology 35,766-770 (2001) I i 000062