ultra-sensitive detection of pharmaceutical and personal...

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C h e m i c a l a n d A p p l i e d M a r k e t s

Ultra-sensitive Detection of Pharmaceutical and Personal Care Products (PPCPs) in Water with an

Integrated On-Line Extraction-UHPLC-MS/MS System

Helen Sun, Ph.D.

Bruker Corporation,

Chemical and Applied Markets (CAM)



Pharmaceutical and Personal Care Products (PPCPs)

PPCP category: • Prescription medicine • Over-the-counter drugs • Veterinary drugs • Fragrances • Cosmetics • Sun-screen products • Diagnostic reagents • Nutraceuticals (e.g., Vitamins)



Examples of PPCPs

PPCP name Classification Ibuprofen Analgesic

Gemfibrozil Antilipemic

Cimetidine Anti-acid reflux

Acetaminophen Antipyretic, Analgesic

Thiabendazole Fungicide and parasiticide

Sulfadimethoxine Sulfonamide antibiotic

Caffeine Stimulant

Bisphenol A Plasticizer

Cholesterol Sterol

Testosterone Sex Hormone

Estrogen Sex Hormone

Benzylecgonine Cocaine metabolite, drug of abuse



Source of PPCPs



PPCPs: impact to the planet

Potential risk to environment

Potential risk to ecological system

Potential risk to human

Potential risk to water resource



Monitoring PPCPs in water

Ternes, Thomas A. Human Pharmaceuticals, Hormones And Fragrances: The Challenge of Micropollutants in Urban Water Management, IWA Publishing

Levels of PPCPs detected in the treated municipal waste water

Analytical challenges for PPCP monitor in water: • Trace amount of analyte • Multi-residue analysis

It requires a sensitive instrument with high speed analysis capability



LC-MS/MS for PPCP analysis in water

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200

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300

2007 2008 2009 2010 2011 2012

Published PPCP studies by LC-MS/MS



LC-MS/MS for PPCP quantitation

targeted analysis, high accuracy

MRM mode of LC-MS/MS for quantitation



Sample preparation for PPCP analysis in water

Pre-concentration step: improve sensitivity

Offline solid phase extraction • Labor intensive • High consumable cost

HPLC online extraction • Automatic, less labor intensive • Two pump systems, complicates

plumbing diagram



EVOQ LC-TQ online extraction (OLE)

• On-Line Extraction (OLE) • Binary + Loading pump • Same foot-print

• Bruker Advance™ UHPLCOLE: The first UHPLC with an integrated third pump for online extraction

• Maximum Performance and Flexibility

• OLE Applications: • Sample online pre-concentration • Dual trap capability • Matrix elimination



Bruker LC-TQ Advantage

Advance HPLC, UHPLC (15,000 psi), & UHPLC-OLE (3 pumps in same cabinet) configuration

with built-in column oven and low dead-volume (60uL)

Industry work-horse PAL auto-sampler Vacuum insulated probe - Heated ESI

(VIP-HESI) with Active Exhaust for high-flow sensitivity & robustness

Dual inter-laced Ion funnels for sustained high-sensitivity

Lens-free collision cell design for maximum productivity

Detector technology for fast pos/neg switching & excellent negative ion

performance

Designed for a singular purpose – to reliably quantify thousands of real samples in the fastest ‘sample-to-report’ time possible



The Bruker LC-TQ Advantage Food Testing –Clenbuterol as a contaminant in meat

• Clenbuterol is a sympathomimetic amine (beta-2 agonist). • Side-effect is an increases in basal metabolism rates, which

helps build muscle mass, while reducing body-fat. • Major outbreak in China which sickened over 900 people • Over 17 livestock houses closed in Mexico due to 100% of

samples testing positive for clenbuterol

• EVOQ LC-TQ Advantage • Turn-key method on the EVOQ LC-TQ • Sustained high sensitivity –proof statement available • Available in Compound Based Screening library Day 1

Day 2

Day 3

Day 4



Advance UHPLCOLE Pump configuration: Single Trap

Waste

CTC

MS

Column Oven

A B

Wash In

Wash Out

A B Solvent A

Solvent B

Mixer Purge valve

Purge out

OLE solvent

Purge valve

Waste

1

2

3 4

5

6

1

2

3

4

5 6

7

8

9

10

Maximum Pressure: 100 Mpa/1000 bar/15,000 psi

OLE loading mode



Advance UHPLCOLE Pump configuration: Single Trap

Waste

CTC

MS

Column Oven

A B

Wash In

Wash Out

A B Solvent A

Solvent B

Mixer Purge valve

Purge out

OLE solvent

Purge valve

Waste

1

2

3 4

5

6

1 2

3

4

5 6

7

8

9

10

Maximum Pressure: 100 Mpa/1000 bar/15,000 psi

OLE analyzing mode



Waste

CTC

MS

Column Oven

A B

Wash In

Wash Out

A B Solvent A

Solvent B

Mixer Purge valve

Purge out

OLE solvent

Purge valve

1

2

3 4

5

6

Waste

1

2

3

4

5 6

7

8

9

10

Advance UHPLCOLE Pump configuration: Dual Trap

Analyzing Trap 1 Loading Trap 2



Waste

CTC

MS

Column Oven

A B

Wash In

Wash Out

A B Solvent A

Solvent B

Mixer Purge valve

Purge out

OLE solvent

Purge valve

1

2

3 4

5

6

Waste

1 2

3

4

5 6

7

8

9

10

Advance UHPLCOLE Pump configuration: Dual Trap

Loading Trap 1 Analyzing Trap 2



EVOQ OLE – lowering detection limit



EVOQ OLE application – PPCP quantitation in city tap water samples and river water



PPCP compounds Category Alprazolam anxiolytic

Ciproxacin antibiotic

Sulfamethoxazole antibiotic

Trimethoprim antibiotic

Carbamazepine anticonvulsant

2,4-D pesticide/herbicide

Ametryn pesticide/herbicide

Atrazine pesticide/herbicide

Bentazone pesticide/herbicide

Carboxine pesticide/herbicide

Cyanazine pesticide/herbicide

Diuron pesticide/herbicide

Hexazinone pesticide/herbicide

Hydroxy Atrazine pesticide/herbicide

MCPA pesticide/herbicide

Mecoprop pesticide/herbicide

Metolachlor pesticide/herbicide

Metribuzin pesticide/herbicide

Prometryn pesticide/herbicide

Pyriproxyfen pesticide/herbicide

Simazine pesticide/herbicide

Thiabendazole pesticide/parasiticide

Dapoxetine prescription drug

Sildenafil prescription drug

Caffeine stimulant

PPCP list



Experimental condition

• LC

OLE pump • Flow rate: 500 µL/min • Gradient: isocratic, 100% H2O + 0.1% FA • Column: YMC-Pack Pro ODS-AQ,

35mm × 2.0 mm, 3 µm • Injection volume: 400 µL

UHPLC binary pump • Flow rate: 400 µL/min • Solvent A: H2O + 5mM NH4OAc • Solvent B: MeOH • Column: YMC-Pack Pro C18 RS, 50mm × 2.0 mm, 3 µm • Gradient run: 1 5 min

• MS condition • Bruker Advance UHPLCOLE

Instrument • Bruker EVOQ Elite

Ion source condition

• HESI: ±4000 V • Cone gas flow: 15 unit • Cone temp: 300 °C • Heated Probe gas: 50 unit • Heated Probe temp: 450 °C • Nebulizer gas flow: 50 unit • Exhaust gas: on

• Sample preparation • Water sample acidified to

0.1% FA and filtered (0.45µm)



Selected PPCPs at 2 ppt



Linearity, 1 ppt – 100 ppt

Trimethoprim

R2=0.999

Carbamazepine R2=0.999

Naproxen R2=0.999

Triclosan

R2=0.998

Gemfibrozil

R2=0.997

Sulfamethoxazole R2=0.999



Summary of PPCPs in Water

Compound Name

RT (min)

ESI R2

S/N (p/p) 2 ppt

RSD% n=10

(5ppt) Tap Water 1 Tap Water 2

Creek Water

Bottled Water

pg/mL (ppt) Trimethoprim 1.66 + 0.994 389 5.80% <2 <2 5 <2

Hydroxy Atrazine 1.88 + 0.997 61 9.20% 4 <2 7 <2 Thiabendazole 1.93 + 0.996 408 8.70% ND <2 <2 <2

Ciproxacin 2.03 + 0.999 25 4.90% ND ND ND ND Caffeine 2.19 + 0.995 68 8.00% ND <2 <2 10 Sildenafil 2.33 + 0.998 132 6.60% ND ND ND <2

Sulfamethoxazole 2.51 + 1 68 5.00% <2 <2 ND <2 Cyanazine 4.1 + 0.998 201 8.70% ND ND ND <2 Simazine 4.38 + 1 56 9.40% 3 <2 5 ND

Metribuzin 4.39 + 0.999 26 12.50% ND ND ND ND Hexazinone 4.42 + 0.997 553 4.90% 17 3 3 ND Dapoxetine 4.62 + 0.999 268 6.00% ND ND ND ND Bentazone 4.69 - 0.999 360 6.20% ND ND ND ND Ametryn 4.73 + 0.997 404 5.70% ND ND <2 ND

Carboxine 4.8 + 0.999 217 6.20% ND ND ND ND Carbamazepine 4.85 + 0.999 485 4.50% <2 <2 <2 ND

Atrazine 5.2 + 0.996 39 6.90% <2 ND ND ND Alpazolam 5.38 + 0.998 178 5.90% ND ND ND ND

Diuron 5.54 + 0.999 56 3.60% 9 <2 6.2 ND Prometryn 5.56 + 0.994 376 8.40% ND ND ND <2

2,4-D 5.7 - 1 26 5.60% 9 <2 13 <2 MCPA 5.81 - 1 56 3.80% <2 <2 <2 ND

Mecoprop 6.39 - 1 27 2.90% <2 <2 11 2 Metolachlor 6.76 + 0.996 177 9.20% 22 <2 <2 <2 Pyriproxifen 8.36 + 1 23 3.50% ND <2 ND <2



Hexazinone

• Broad spectrum herbicide • Inhibits photosynthesis • Used to control grasses,

wood plants • High water solubility • A pervasive groundwater

contaminant



Metolachlor

• Chloroacetanilide herbicide • Has been detected in ground

and surface water through out US (0.08 – 4.5 ppb)

• Category C pesticide by US EPA • Bioaccumulation of

metolachlor in edible species of fish

• US EPA health advisory level (HAL): 0.525 ppm



Summary

• EVOQ OLE system provides a total solution for trace PPCP quantitation in water samples: automatic online extraction followed by LC-MS/MS targeted analysis

• An MRM based LC-MS/MS method has been developed for PPCP quantitation in city water, creek water and bottled water samples

• Quantitation result shows that the water quality between cities is slightly different, weed killer herbicides were screened in the tested water samples, but the concentration is still within safety range

• EVOQ OLE system is a ease-of-use, sensitive and robust instrument for PPCP analysis in water samples



Conclusion

GC-TQ or LC-TQ MRM Data Reports

Data Generation Information Creation Decisions

Game-Changing Products for Quadrupole MS Applications

ultra-sensitive detection of pharmaceutical and personal...

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