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Agilent Optimizing Detection of Steroids in Wastewaters Using the Agilent 6490 Triple Quadrupole LC/MS System with iFunnel Technology Manual

1. 0 1 pg L std 10 pg on column 100 pg L Figure 6 82 84 86 88 90 92 94 96 98 100 102 Acquisition time min Detection of 1 pg to 100 pg on column of estrone calibration standard using the optimized method parameters Postcolumn addition of 0 1 ammonia solution at 0 1 mL min also gives an increase in response for the steroids when using the optimal MS parameters in the order of 3 4 fold compared to no addition Figure 7 x108 7 04 Estrone 1 8 fold increase _ Postcolumn addition of 0 1 NH Estradiol 3 7 fold increase 8 100 Counts w eT ag m Ethynyl estradiol 3 1 fold increase No postcolumn addition 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 Acquisition time min Figure 7 Postcolumn addition of 0 1 ammonia can increase response by as much as almost 4 fold for all three steroids analyzed Sensitive Detection of Steroids in Sewage Effluent This optimized method can detect sub part per trillion ng L steroids in wastewater Figure 8 shows ethynyl estradiol at 0 91ng L in crude sewage as well as the excellent linearity of the calibration curve R2 gt 0 9999 Estrone was easily detectable at 53 7 ng L Figure 9 in crude sewage and estradiol at 44 5 ng L Figure 10 Both of these steroids also showed excellent linearity of quantification with R2 values gt 0 9999 37132011 537 PM a ae SSPN pE m TYAN 729 PM a 24S
2. 40 C 25 uL A Water B Acetonitrile 16 0 min 0 3 mL min 0 1 mL min postcolumn addition of 0 1 NH Time min Solvent A Solvent B 0 90 10 0 5 60 40 10 0 20 80 10 2 0 100 11 5 0 100 11 6 90 10 ESI mode negative ionization Dynamic MRM 300 C 11 L min 180 C Nitrogen 16 L min 45 psig 1 500 V 3 000 V Varied per optimization study Varied per optimization study Varied per optimization study Delta EMV 300 V The Agilent Triple Quadrupole LC MS System analysis parameters are shown in Table 2 Table 2 Time Segments Transitions and Fragmentor Voltages used for Optimal Analysis of the Steroid Compounds Retention time Time segment min 1 8 0 1 8 0 1 8 5 1 8 5 1 8 8 1 8 8 Precursor Product Fragmentor Compound ion ion CAV voltage Estradiol 271 0 145 0 2 380 Estradiol d5 276 0 147 0 2 380 EthynylEstradiol 295 0 145 0 2 380 EthynylEstradiol d4 299 0 147 0 2 380 Estrone 269 0 145 0 2 380 Estrone d4 273 0 147 0 2 380 Collision energy 50 50 52 52 45 45 Results and Discussion Optimization of Analysis Parameters While methods exist for steroid analysis on previous instru ments the analysis parameters for these methods must be optimized in order to take full advantage of the unique sensi tivity of the Agilent 6490 Triple Quadrupole LC MS with iFunnel Technology 6490 In this case the method used as a starting point was developed on the 6460 Triple Quadrupole LC MS 6460 Col
3. min Figure 2 Significant increase in response due to increasing the multiplier voltage Delta EMV x10 Counts 50 V Opt 6490 CE voltage Opt 35 50 V 35 V Opt 6460 Figure 3 7 6 7 7 7 8 7 9 8 0 8 1 8 2 8 3 8 4 8 5 8 6 8 7 8 8 8 9 9 0 9 1 9 2 9 3 9 4 Acquisition time min Figure 4 78 79 80 Acquisition time min 77 Optimization of collision energy CE on the Agilent 6490 Triple Quadrupole LC MS System provides an increase in response for detection of estradiol of almost two fold compared to the collision energy that was optimal on the 6460 35 volts 60 V default Optimization of collision energy CE is also critical for maxi mizing response While the optimal collision energy for the 6460 method was 35 volts for estradiol increasing the voltage in 5 volt increments on the 6490 continually increased response with a nearly two fold increase at 50 volts Figure 3 One of the keys to the enhanced sensitivity of the Agilent 6490 Triple Quadrupole LC MS System is the proprietary Agilent iFunnel Technology which utilizes a novel dual stage ion funnel assembly Both the low and high pressure voltage have default settings in the tune file of the 6490 However both voltages can be optimized to significantly increase sensi tivity For example changing the low pressure voltage from the default of 60 volts to 100 volts can increase response by as much as 65 as can changing the hi
4. with 4 mL of ethyl acetate The extract was evaporated under a gentle air stream with the vial on a 45 C heating block and the residue redissolved in 250 uL of cyclohexane propan 2 ol 95 5 The extracts were cleaned up using normal phase chromatog raphy on an Agilent 1100 series LC with fraction collection on an Agilent ZORBAX Cyano column p n 883952 705 4 6 x 150 mm 5 pm thermostated at 55 C using an isocratic separation 95 5 cyclohexane propan 2 ol at a flow rate of 1 mL min fraction collection time range 5 8 8 6 minutes The extract was evaporated under a gentle air stream with the vial on a 45 C heating block and the residue re dissolved in 250 uL of methanol water 90 10 With a final volume of 250 uL this is equivalent to a 1000 fold concentration step Therefore the calibration standards are equivalent to original concentrations of 1 2 5 and 10 ng L Table 1 LC and MS Instrument Conditions LC Conditions Analytical column Guard column Column temperature Injection volume Mobile phase Run time Flow rate Gradient program MS Conditions Acquisition parameters Sheath gas temperature Sheath gas flow rate Gas temperature Drying gas Nebulizer pressure Nozzle voltage Vcap voltage Cell accelerator voltage Delta EMV Low and high pressure ion funnel voltage Scan type dynamic MRM Analysis Parameters Agilent C 18 Eclipse Plus 2 1 x 50 mm 3 5 um p n 959763 902 Luna C 18 4 0 mm x 2 0 mm
5. Author Neil Cullum Anglian Water Services Huntingdon England Optimizing Detection of Steroids in Wastewaters Using the Agilent 6490 Triple Quadrupole LC MS System with iFunnel Technology Application Note Environmental Abstract Taking full advantage of the enhanced sensitivity of the Agilent 6490 Triple Quadrupole LC MS System requires optimization of method parameters derived on other Triple Quadrupole platforms Optimization of steroid analysis methods in the complex matrix of wastewater results in sensitivity as low as sub 1 ng L 1 part per trillion Introduction Pharmaceuticals are consumed in high quantities worldwide and these amounts will continue to increase due to improving health care and longer life expectations Administered pharmaceuticals are excreted by humans as a parent compound or metabolites They enter sewage treatment plants where they are not entirely removed and end up in the environment These residual pharmaceuticals can negatively impact aquatic and terrestrial ecosystems A recent study by the United States Geological Survey USGS found chemical cont aminants in 80 of the streams sampled and steroids were one of the chemical groups most frequently detected These steroids elicit biological responses at very low concentrations including feminization of male fish A seven year whole lake experiment in Canada demonstrated that chronic exposure of a fish species to the steroid 17 alpha ethynyl e
6. PM sis aa BMP EA me ial et 7132011 22 PM soj pasaj sot aat et Ta EGEJ 132011 938 PM 100 an Ee elton asses ST T E E T T E E E sa 11306140 ina5a Sampie msamaa 100 es 8m ossea assz agai 15773 Cla linasa sere name 10 asal aaron taal rsl an 21 re J J6 38 2 d2 da d6 as 82 Se Relative Concentration veneer Figure 8 Analysis of 0 91 ng L of ethynyl estradiol in crude sewage showing the MRM chromatogram as well as the chromatogram for the internal standard and the calibration curve with an R value gt 0 9999 Pa a a aa em SEAE paneas foj pam ae Figure 9 Analysis of 53 7 ng L of estrone in crude sewage showing the MRM chromatogram as well as the chromatogram for the internal standard and the calibration curve with an R value gt 0 9999 Esro oi Esval Renta Esvasot0s den Deine a a oS Sse cane fe eae 732011832 PM 20762 Ovi 17358 i anism sexe st tose a ana 7A320150 PM eet oso sista toccos 100005 1o00 a011 16520 21888 213875 pansa 5I PM 100 8 050 em 50 80sof 8113 Tiny firmos ijod Sampie nyama S0 ao EEA Pu T S TANYA 657 342011 7 13 PM af Samgle 732013 7 45 PM 100 Surele Sn 7132011 801 PM ne moe S18 PM rel mamani sot fa k e ae iaa 850 PM a 9 06 PM 7032011922 PM 7032011 9 38 PM 7132011 954 PM __
7. gh pressure high RF voltage from the default of 150 volts to 160 volts Figure 4 Applying all of these optimized parameters to the method on the 6490 results in response that is as much as three times higher than that obtained from these steroids using the opti mized parameters from the 6460 method on the 6490 Figure 5 Applying these optimized parameters enables the detection of as little as 0 1 pg of estrone Figure 6 estradiol and ethynyl estradiol calibration standards on the column 10 pg L in the sample 2 14 B 2 64 25 High pressure ion funnel RF 8 1 8 3 85 87 89 Acquisition time min 9 1 Optimization of both the low pressure and high pressure ion funnel voltages on the Agilent 6490 Triple Quadrupole LC MS System provides an increase in response of as much as 65 for both settings for the three steroids lon funnel optimized CE and CAV optimized 6460 CE default CAV No optimization Figure 5 Counts gt co i 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 J4 95 Acquisition time min The cumulative effect of the various Agilent 6490 Triple Quadrupole LC MS System optimization steps on response is shown resulting in as much as much as three times higher response than that obtained from these steroids using the optimized parameters from the Agilent 6490 Triple Quadrupole LC MS System method Estrone 6490 opt conditions 1 ug L std 100 pg on column 1 000 pg L
8. imental Reagents and Standards The ethyl acetate acetonitrile propan 2 ol cyclohexane and methanol were all HPLC grade or glass distilled The hydrochloric acid was 37 analytical reagent grade and ammonia solution 30 The copper nitrate was general pur pose grade reagent or better The water was HPLC grade or Elga polished water The styrene divinyl benzene solid phase extraction SPE cartridges 200 mg were obtained from Baker The GF D glass microfiber filter papers were obtained from Whatman Kent UK Individual solutions of 100 mg L of each steroid estrone estradiol and ethynyl estradiol in acetonitrile were obtained from QMX Laboratories Ltd Thaxted UK A mixed calibration standard was prepared by adding each of the individual steroid solutions to methanol to a concentration of 1 0 mg L Working calibration standards were prepared by dilution in 90 10 water methanol to 1 2 5 and 10 pg L These calibra tion standards also contained 2 ug L of each internal standard Solid deuterated estrone D4 estradiol D5 and ethynyl estra diol D4 were obtained from QMX Laboratories for use as internal standards Individual 100 mg L solutions of each internal standard were prepared in acetronitrile then a mixed internal standard was prepared by adding each internal standard to methanol to a final concentration of 1 0 mg L A second mix was prepared by diluting an aliquot of the 1 0 mg mL mix to 0 1 mg L with methanol Ins
9. lision cell accelerator voltage CAV is a key parameter in optimizing production detection and quantitation of ions Figure 1 illustrates the significant increase in response for detection of the three steroids prepared as calibration stan dards obtained by changing the accelerator voltage from 5 to 2 volts x10 4 6 4 4 4 24 4 04 38 Cell acc voltage 2 versus 5 3 64 3 44 3 24 3 04 2 8 2 6 2 4 2 24 2 05 1 8 1 6 1 45 1 27 1 0 Estrone Be ee Estradiol Counts Ethynyl estradiol H T T T T T T T T ji T T T T T I 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 Acquisition time min Figure 1 Effect on response of changing the cell accelerator voltage CAV from 5 volts lower peaks to 2 volts higher peaks The multiplier voltage Delta EMV is another parameter that significantly influences response By varying the voltage from 100 V to 400 V in 100 V increments the response is increased by as much as a factor of 14 across the range In excess of 400 volts the response will still increase but the signal to noise ratio can stay static or indeed decrease The optimized value was found to be around 200 300 volts Figure 2 6 6 Estrone Estradiol Counts SS 292 SS NNN CC ea ea e a nA Nne SCYERBDONWERDONEROONARROONEROONEROON P Ethynyl Estradiol T T T T T T T T T T T T T T T T T 75 7 6 7 7 78 7 9 8 0 81 82 83 84 85 86 87 88 89 9 0 9 1 92 Acquisition time
10. ludge pdf slu dge_pollutants_xsum pdf For More Information These data represent typical results For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2012 Printed in the USA February 24 2012 5990 9978EN j Agilent Technologies
11. soso me 28201 TAIZ TOON 100 3033 l 2 0082 See manan sensu arer Serele 7na2oir1azem 10 A MAM 276 0 gt 147 0 tAd Smoot 1 Figure 10 Analysis of 44 5 ng L of estradiol in crude sewage showing the MRM chromatogram as well as the chromatogram for the internal standard and the calibration curve with an R value gt 0 9999 Conclusions The Agilent 6490 Triple Quadrupole LC MS System with iFunnel Technology provides exquisite sensitivity making it the ideal choice for applications that require the detection of minute quantities of analyte However as with any other instrument methods that have been developed on earlier gen eration MS instruments must be optimized on the 6490 in order to maximize sensitivity Such optimization can increase the response for detection of steroids by as much as three times versus the response obtained when using the parame ters taken from the method developed on the Agilent 6460 Triple Quadrupole LC MS System The optimized method can provide sub ng L detection of steroids in wastewaters which are very complex matrices References 1 K A Kidd P J Blanchfield K H Mills V P Palace R E Evans J M Lazorchak R W Flick Collapse of a fish population after exposure to a synthetic estrogen Proc Natl Acad Sci U S A 104 8897 8901 2007 Pollutants in Urban Waste Water and Sewage Sludge http ec europa eu environment waste s
12. stradiol led to a near extinction of that species from the lake 1 There is also potential for these steroids to travel up the food chain as well as into drinking water Agilent Technologies The European Union Water Framework Directive 2000 60 EC 2 promotes sustainable water use including the long term reduction of wastewater contaminant dis charges to the aquatic environment including steroids Implementation of the Directive requires the development of sensitive accurate and reliable testing methods This application note describes the optimization of sensitive methods for steroid detection on the Agilent 6490 Triple Quadrupole LC MS System with iFunnel Technology as part of the Chemical Investigation Program of Directive 2000 60 EC This instrument takes detection limits lower than ever enabling zeptomole level sensitivity at conventional flow rates making it an ideal choice for critical pharmaceuti cal applications including the detection of steroids in the environment While liquid chromatography mass spectrome try LC MS methods for steroid detection and quantification have been developed on earlier generation MS instruments they must be optimized on the 6490 Triple Quadrupole LC MS System in order to maximize sensitivity Using the optimal parameters determined for detection of several steroid com pounds detection limits as low as sub 1 ng L one part per trillion in wastewaters have been demonstrated Exper
13. truments Method optimization and analysis of wastewaters were con ducted on the Agilent 1260 Infinity LC with a 100 uL sample loop coupled to the 6490 Triple Quadrupole LC MS System with iFunnel Technology Postcolumn addition of 0 1 ammo nia solution was accomplished using an external pump attached to a T junction between the column and the nebu lizer The instrument conditions are listed in Table 1 Sample Collection Preparation and Cleanup Samples were collected in 2 L amber glass bottles and pre served at the time of sampling with 2 mL of concentrated hydrochloric acid and 0 5 g of copper nitrate and stored at below 10 C Sample stability under these conditions has been tested up to 14 days after preservation Once extraction has taken place the resulting extracts can be stored for at least four weeks in a spark proof refrigerator prior to analysis Each sample was filtered through GF D papers prior to extrac tion An aliquot of 20 uL of mixed internal standard was added to 1 L of sample For crude sewage samples 100 mL of sample was diluted with 900 mL of water and 20 uL of mixed internal standard was added The sample was then extracted with the SPE cartridge which was first conditioned with ethyl acetate 5 mL then methanol 5 mL then water 3 mL The cartridge was loaded with 250 mL of sample then washed with 60 methanol 3 mL followed by water 3 mL After drying with gas for 40 minutes the cartridge was eluted

Agilent Optimizing Detection of Steroids in Wastewaters Using the Agilent 6490 Triple Quadrupole LC/MS System with iFunnel Technology Manual

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