EPA Method 1694: Agilent's 6410A

LC/MS/MS Solution for

Pharmaceuticals and Personal Care
Products in Water, Soil, Sediment,
and Biosolids by HPLC/MS/MS
Application Note

Environmental

Authors Abstract
Imma Ferrer and E. Michael Thurman An analytical methodology for screening and confirming the presence of 65 pharma-
Center for Environmental Mass ceuticals in water samples was developed using the Agilent G6410A Triple
Spectrometry Quadrupole mass spectrometer (QQQ). The method was developed following the
University of Colorado guidelines in EPA Method 1694. Four distinct chromatographic gradients and LC con-
Civil, Environmental, and Architectural ditions were used according to the polarity and extraction of the different pharmaceu-
Engineering
ticals. Positive and negative ion electrospray were used with two multi-reaction moni-
ECOT 441, 428 UCB
toring (MRM) transitions (a quantifier and a qualifier ion for each compound), which
Boulder, CO 80309
USA adds extra confirmation in this methodology compared with the EPA method. Linearity
of response of three orders of magnitude was demonstrated (r2 > 0.99) for all the
Jerry Zweigenbaum
pharmaceuticals studied. The analytical performance of the method was evaluated for
Agilent Technologies, Inc.
one wastewater sample collected from Boulder Creek, Colorado; positive identifica-
2850 Centerville Road
tions for carbamazepine and diphenhydramine were found for this sample using the
Wilmington, DE 19808
methodology developed in this work.
USA
Introduction tandem mass spectrometer using a single transition for each
compound. This application note describes the Agilent solu-
The analytical challenge of measuring emerging contaminants tion to this method, which is demonstrated with the Agilent
in the environment has been a major research focus of scien- model 6410A LC/MS QQQ. The Agilent initial implementation
tists for the last 20 years. Pharmaceuticals and personal care for EPA Method 1694 consists of 65 analytes (of 75 total ana-
products (PPCPs) are an important group of contaminants lytes) and 17 labeled internal standards (of 20 total), which
that have been targeted, especially in the last decade. In the are a mixture of PPCPs that are analyzed each by a single
area of PPCPs there are several methods addressing the MRM transition. (Note that the other compounds and internal
analysis of these analytes, including EPA Method 1694 [1], standards could not be obtained at this time.) The method
which was recently published (December 2007). This EPA pro- also uses Agilent C-18 and Hydrophilic Interaction
tocol uses solid-phase extraction (SPE) for water sample Chromatography (HILIC) columns for all analytes. To provide
preparation [1]. The extracts are then analyzed directly by a additional confirmation, a second MRM transition was added
for 60 of the 65 analytes analyzed. This gives an even greater
assurance of correct identification than prescribed by the
EPA. Table 1 shows the list of pharmaceuticals studied here.

Table 1. Analytes Studied in This Work

List of Group 1 Compounds EPA 1694: 46 Analytes

Acetaminophen Codeine Flumequine Penicillin V Sulfanilamide
Ampicillin Cotinine Fluoxetine Roxithromycin Thiabendazole
Azithromycin Dehydronifedipine Lincomycin Sarafloxacin Trimethoprim
Caffeine Digoxigenin Lomefloxacin Sulfachloropyridazine Tylosin
Carbadox Diltiazem Miconazole Sulfadiazine Virginiamycin
Carbamazepine 1,7-Dimethylxanthine Norfloxacin Sulfadimethoxine Digoxin*
Cefotaxime Diphenhydramine Ofloxacin Sulfamerazine
Ciprofloxacin Enrofloxacin Oxacillin Sulfamethazine
Clarithromycin Erythromycin Oxolinic acid Sulfamethizole
Cloxacillin Erythromycin anhydrate Penicillin G Sulfamethoxazole
*Compound formed intractable Na adduct with current conditions.

List of Group 2, 3, and 4 Compounds: EPA 1694: 19 Analytes

Anhydrotetracycline (2) Doxycycline (2) Minocycline (2) Triclocarban (3)
Triclosan (3)
Warfarin (3)
Chlorotetracycline (2) 4-Epianhydrotetracycline (2) Tetracycline(2) Albuterol (4)
Meclocycline (2) Cimetidine (4)
Metformin (4)
Demeclocycline(2) 4-Epitetracycline(2) Gemfibrozil (3) Ranitidine (4)
Ibuprofen (3)
Naproxen (3)

List of Labeled Internal Standards

13C -15N-Acetaminophen 13C -Erythromycin 13C -Sulfamethazine 13C -Trimethoprim
2 2 6 3
13C -Atrazine Fluoxetine-d6 13C -Sulfamethoxazole Warfarin-d5
3 6
13C -Caffeine Gemfibrozil-d6 13C -2,4,5-Tricloro- Carbamazepine-d10
3 6
phenoxyacetic acid (Extra compound, not EPA list)
13C -15N-Ciprofloxacin 13C -Ibuprofen 13C -Triclocarban
3 3 6
Cotinine-d3 13C-Naproxen-d 13C -Triclosan
3 12

2
Experimental LC conditions for Group 2-acidic extraction, positive electrospray
ionization (ESI+) instrument conditions
Sample Preparation
Column Agilent ZORBAX Eclipse Plus C18
Pharmaceutical analytical standards were purchased from 2.1 × 100 mm, 3.5 µ (p/n 959793-902)
Sigma, (St. Louis, MO). All stable isotope labeled compounds Column temperature 25 °C
used as internal standards were obtained from Cambridge Mobile phase 10% ACN and 90% H2O with 0.1% HCOOH
Isotope Laboratories (Andover, MA). Individual pharmaceuti-
Flow rate 0.2 mL/min
cal stock solutions (approximately 1,000 µg/mL) were pre-
pared in pure acetonitrile or methanol, depending on the solu- Gradient t0 = 10% ACN
bility of each individual compound, and stored at t10 = 10% ACN
t30 = 100% ACN
–18 °C. From these solutions, working standard solutions
were prepared by dilution with acetonitrile and water. Injection volumes 15 µL

Water samples were collected from the wastewater treat-

ment plant at the Boulder Creek outfall (Boulder, CO) and LC conditions for Group 3-acidic extraction, negative electrospray
extracted as per the EPA method. Agilent has introduced a
ionization (ESI–) instrument conditions
polymeric SPE sorbent with hydrophilic/lipophilic properties
that may also be appropriate for this application. “Blank” Column Agilent ZORBAX Eclipse Plus C18
wastewater extracts were used to prepare the matrix- 2.1 × 100 mm, 3.5 µ (p/n 959793-902)
matched standards for validation purposes. The wastewater Column temperature 25 °C
extracts were spiked with the mix of pharmaceuticals at dif- Mobile phase 40% MeOH and 60% H2O with
ferent concentrations (ranging from 0.1 to 500 ng/mL or ppb) 5 mM ammonium acetate, pH 5.5
and subsequently analyzed by LC/MS/MS.
Flow rate 0.2 mL/min
LC/MS/MS Instrumentation Gradient t0.5 = 40% MeOH
t7 = 100% MeOH
The analytes were subdivided in groups (according to EPA
protocol for sample extraction) and LC conditions for the Injection volumes 15 µL
chromatographic separation of each group are as follows.
LC conditions for Group 4-acidic extraction, positive electrospray
LC Conditions for Group 1-acidic extraction, positive ionization (ESI+) instrument conditions
electrospray ionization (ESI+) instrument conditions
Column Agilent ZORBAX HILIC Plus
Column Agilent ZORBAX Eclipse Plus C18 2.1 × 100 mm, 3.5 µm (p/n 959793-901
2.1 × 100 mm, 3.5 µ (p/n 959793-902) custom order until November 1, 2008)

Column temperature 25 °C Column temperature 25 °C

Mobile phase 10% ACN and 90% H2O with 0.1% HCOOH Mobile phase 98% ACN and 2% H2O with 10 mM
ammonium acetate, pH 6.7
Flow rate 0.2–0.3 mL/min
Flow rate 0.25 mL/min
Gradient t0 = 10% ACN, 0.2 mL/min
t5 = 10% ACN, 0.2 mL/min Gradient t0 = 98% ACN
t6 = 10% ACN, 0.3 mL/min t5 = 70% ACN
t24 = 60% ACN, 0.3 mL/min t12 = 70% ACN
t30 = 100% ACN Injection volumes 15 µL
Injection volumes 15 µL

3
The mass spectrometer conditions were general to all groups Results and Discussion
and are as follows.
Optimization of LC/MS/MS Conditions
MS Conditions
Mode Positive and negative (depending on The initial study consisted of two parts. First was to optimize
group) ESI using the Agilent G6410A the fragmentor voltage for each of the pharmaceuticals stud-
Triple Quadrupole mass spectrometer ied in order to produce the largest signal for the precursor ion.
Nebulizer 40 psig Typically the protonated molecule was used for the precursor
Drying gas flow 9 L/min ion. Each compound was analyzed separately using an auto-
mated procedure (MassHunter Optimizer software, Agilent
V capillary 4000 V
Technologies, Santa Clara, CA) to check the fragmentor at
Drying gas temperature 300 °C each voltage. The data was then selected for optimal frag-
Fragmentor voltage 70–130 V mentor signal and each compound was optimized again to
Collision energy 5–35 V determine automatically the collision energies for both the
MRM 2 transitions for every compound as shown quantifying and qualifying ions. Optimal collision energies var-
in Table 1 ied between 5 and 35 V. The MRM transitions and optimized
energies used for this study are shown in Tables 2A to 2D.
Dwell time 10 msec

Table 2A. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 1 (The
labeled standards are bold.)

Fragmentor MRM Collision energy

Compound voltage transitions (m/z) (eV)
Acetaminophen 90 152 → 110 15
152 → 65 35
13C -15N-Acetaminophen
2 90 155 → 111 15
155 → 93 25
Ampicillin 70 350 → 160 10
350 → 106 15
13C -Atrazine
3 120 219 → 177 15
219 → 98 25
Azithromycin 130 749.5 → 591.4 30
749.5 → 158 35
Caffeine 110 195 → 138 15
195 → 110 25
13C -Caffeine
3 110 198 → 140 15
198 → 112 25
Carbadox 80 263 → 231 5
263 → 130 35
Carbamazepine 110 237 → 194 15
237 → 179 35
Carbamazepine-d10 110 247 → 204 15
247 → 202 35
Cefotaxime 90 456 → 396 5
456 → 324 5
Ciprofloxacin 110 332 → 314 20
332 → 231 35
13C -15N-Ciprofloxacin
3 110 336 → 318 15
336 → 235 35

4
Table 2A. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 1
(The labeled standards are bold.) continued

Fragmentor MRM Collision energy

Compound voltage transitions (m/z) (eV)

Clarithromycin 110 748.5 → 158 25

748.5 → 590 15
Cloxacillin 90 436 → 160 15
436 → 277 15
Codeine 130 300 → 215 25
300 → 165 35
Cotinine 90 177 → 98 25
177 → 80 25
Cotinine-d3 90 180 → 80 25
180 → 101 25
Dehydronifedipine 130 345 → 284 25
345 → 268 25
Digoxigenin 90 391 → 355 15
391 → 337 15
Digoxin No response, Na adduct
Diltiazem 130 415 → 178 25
415 → 150 25
1,7-Dimethylxanthine 90 181 → 124 15
181 → 99 15
Diphenhydramine 70 256 → 167 15
256 → 152 35
Enrofloxacin 130 360 → 316 15
360 → 342 15
Erythromycin 90 734.5 → 158 35
734.5 → 576 15
13C -Erythromycin
2 90 736.5 → 160 25
736.5 → 578 15
Erythromycin anhydrate 90 716.5 → 158 25
716.5 → 116 25
Flumequine 90 262 → 174 35
262 → 244 15
Fluoxetine 90 310 → 148 5
Fluoxetine-d6 90 316 → 154 5
Lincomycin 110 407 → 126 25
407 → 359 15
Lomefloxacin 130 352 → 308 15
352 → 265 25
Miconazole 90 415 → 159 35
415 → 69 25
Norfloxacin 70 320 → 302 15
320 → 276 15
Ofloxacin 110 362 → 318 15
362 → 261 25

5
Table 2A. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 1 (The
labeled standards are bold.) continued
Fragmentor MRM Collision energy
Compound voltage transitions (m/z) (eV)

Oxacillin 70 402 → 160 15

402 → 243 5
Oxolinic acid 90 262 → 244 15
262 → 216 25
Penicillin G 90 335 → 160 5
335 → 176 5
Penicillin V 70 351 → 160 5
351 → 114 25
Roxithromycin 130 837.5 → 679 15
837.5 → 158 35
Sarafloxacin 130 386 → 299 25
386 → 368 25
Sulfachloropyridazine 90 285 → 156 10
285 → 92 25
Sulfadiazine 110 251 → 156 15
251 → 92 25
Sulfadimethoxine 80 311 → 156 20
311 → 92 35
Sulfamerazine 110 265 → 156 15
265 → 92 25
Sulfamethazine 90 279 → 156 15
279 → 186 15
13C -Sulfamethazine
6 90 285 → 186 25
285 → 162 25
Sulfamethizole 80 271 → 156 10
271 → 92 25
Sulfamethoxazole 110 254 → 156 15
254 → 92 25
13C -Sulfamethoxazole
6 110 260 → 162 15
260 → 98 25
Sulfanilamide 70 173 → 156 5
173 → 92 15
Thiabendazole 130 202 → 175 25
202 → 131 35
13C -2,4,5-Trichlorophenoxyacetic
6 acid 110 259 → 201 5
259 → 165 25
Trimethoprim 110 291 → 230 25
291 → 261 25
13C -Trimethoprim
3 110 294 → 233 25
294 → 264 25
Tylosin 110 916.5 → 174 35
916.5 → 772 35
Virginiamycin 110 526 → 508 5
526 → 355 15

6
Table 2B. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 2

Fragmentor MRM Collision energy

Compound voltage transitions (m/z) (eV)

Anhydrotetracycline 90 427 → 410 15

427 → 154 25
Chlorotetracycline 110 479 → 462 15
479 → 197 35
Demeclocycline 130 465 → 430 25
465 → 448 15
Doxycycline 110 445 → 428 15
445 → 154 25
4-Epianhydrotetracycline (EATC) 90 427 → 410 15
427 → 105 35
4-Epitetracycline (ETC) 110 445 → 410 15
445 → 427 5
Minocycline 90 458 → 441 15
Tetracycline (TC) 110 445 → 410 15
445 → 427 5

Table 2C. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 3

Fragmentor MRM Collision energy

Compound voltage transitions (m/z) (eV)
Gemfibrozil 100 249 → 121 5
Gemfibrozil-d6 100 255 → 121 5
Ibuprofen 75 205 → 161 5
13C -Ibuprofen
3 75 208 → 163 5
Naproxen 75 229 → 169 25
229 → 170 5
13C-Naproxen-d
3 75 233 → 169 25
233 → 170 5
Triclocarban 100 313 → 160 10
313 → 126 25
13C -Triclocarban
6 90 319 → 160 5
319 → 132 25
Triclosan 75 287 → 35 5
13C -Triclosan
12 75 299 → 35 5
Warfarin 125 307 → 117 35
307 → 161 15
Warfarin-d5 90 312 → 161 15
312 → 255 25

7
Table 2D. MRM Transitions and MS Operating Parameters Selected for the Analysis of the Pharmaceutical Compounds in Group 4

Fragmentor MRM Collision energy

Compound voltage transitions (m/z) (eV)

Albuterol (Salbutamol) 90 240 → 148 15

240 → 166 5
Cimetidine 100 253 → 159 10
253 → 95 25
Metformin 80 130 → 60 10
130 → 71 25
Ranitidine 110 315 → 176 15
315 → 130 25

Chromatographic separation was done independently for each

group and a dwell time of 10 msec was used for every MRM
transition. Figures 1A to 1D show the chromatograms corre-
sponding to 100 ppb standard on column for all the pharma-
ceuticals studied. Extracted ion chromatograms are overlaid
for each one of the target analytes according to their respec-
tive protonated molecule and product-ion MRM transitions.

×10 3
7.0 1 12 23 3
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Counts vs. acquisition time (min)

Figure 1A. MRM extracted chromatogram for pharmaceuticals in Group 1. Three time segments were used in this chromatographic separation.

8
×10 3 479 → 462
1 1

0
×10 2 465 → 430
1 1

0
×10 2
1
458 → 441 1

×10 3
1
445 → 410 1

×10 3 1
445 → 428 1

0
×10 3
1 427 → 410 1

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Counts vs. acquisition time (min)

Figure 1B. MRM extracted chromatogram for pharmaceuticals in Group 2. Only one transition shown. See Table 2B for compound identification.

×10 4
1 312 → 159.7 1

0
×10 2
1 301 → 116.7 1

0
×10 1
1 287 → 34.6 1

0
×10 3
1 289 → 120.8 1

0
×10 2
1 229 → 168.8 1

0
×10 3
1
205 → 160.9 1

0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12
Counts vs. acquisition time (min)

Figure 1C. MRM extracted chromatogram for pharmaceuticals in Group 3. Only one transition shown. See Table 2C for compound identification.

9
×10 4
3.2 1 1

3 Albuterol
2.8 240 & 166
2.6 & 148
2.4
2.2
2 253 & 159
& 95
1.8
1.6
Cimetidine Metformin
130 & 71
1.4
& 60
1.2
315 & 176
1
& 130
0.8
Ranitidine
0.6
0.4
0.2
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Counts vs. acquisition time (min)

Figure 1D. MRM extracted chromatogram for pharmaceuticals in Group 4.

Application to Wastewater Samples

To confirm the suitability of the method for analysis of real

samples, matrix-matched standards were analyzed in a
wastewater matrix from an effluent site, at eight concentra-
tions (0.1, 0.5, 1, 5, 10, 50, 100, and 500 ng/mL or ppb concen-
trations). Figure 2 shows an example standard curve for
acetaminophen in the wastewater matrix. In general, all com-
pounds gave linear results with excellent sensitivity over
three orders of magnitude, with r2 values of 0.99 or greater.

10
Figure 2. Calibration curve for acetaminophen in a wastewater matrix using a seven-point curve from 0.1 to 100 ng/mL (ppb) using a linear fit with no origin
treatment.

Finally, a “blank” wastewater sample was analyzed and the

presence of two pharmaceuticals, carbamazepine and diphen-
hydramine, could be confirmed with two MRM transitions.
Figure 3 shows the ion ratios qualifying for these two com-
pounds in a wastewater extract. As shown in Figure 3 in the
two ion profiles, both pharmaceuticals were easily identified
in this complex matrix due to the selectivity of the MRM tran-
sitions and instrument sensitivity.

11
 Carbamazepine Diphenhydramine
×10 3
×10 4
23 7 23
3.5
6
3.0
2.5 237 → 194 5 256 → 167
2.0 4
1.5 3
1.0 2
0.5 1
0.0 0
×10 3 ×10 3 2 3
72 3
6 2.5
5 237 → 179 2.0 256 → 152
4 1.5
3
1.0
2
1 0.5
0 0.0
14 15 16 17 18 19 20 21 22 23 24 14 15 16 17 18 19 20 21 22
Counts vs. acquisition time (min) Counts vs. acquisition time (min)

Figure 3. MRM chromatograms of a wastewater sample for carbamazepine and diphenhydramine using two transitions.

Conclusions
The results of this study show that the Agilent 6410A Triple Quadrupole is a robust,
sensitive, and reliable instrument for the study of pharmaceuticals in water samples,
using high throughput methods. The Agilent 6410A Triple Quadrupole has been
shown to be a successful instrument for the implementation of EPA Method 1694.
References
1. EPA Method 1694: Pharmaceuticals and personal care products in water, soil,
sediment, and biosolids by HPLC/MS/MS, December 2007, EPA-821-R-08-002.

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September 19, 2008
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