Fast Screening Method for the Determination and Quantification of Pharmacologically Active Substances

Introduction

Pharmacologically active substances are recognized as an emerging problem for surface water quality and for the handling of waste water plants in Europe. Several researchers have shown that these compounds are able to disturb ecologic balances in surface water (e.g. Kümmerer, K., The presence of pharmaceuticals in the environment due to human use – present knowledge and future challenges. J. Environmental Management 90 (2009) 2354-2366.). Some compounds like Carbamazepine even withstand the biological waste water treatment processes and are found downstream from waste water treatment plants.

Human and animal drugs can be found in the environment either as the drug itself, or as a metabolite/transformation product. More than 100 different pharmacologically active compounds have been identified in surface water in Europe thus far.

During the last revision of the European Water Framework Directive “Directive 2000/60/EC of the European Parliament and of the Council establishing a framework for the Community action in the field of water policy“ this issue was discussed intensively, but only three pharmacologically active substances (the hormonal preparations 17alphaethinylestradiol and 17beta-estradiol and the painkiller Diclofenac) were selected as marker substances and put onto a watch list to gain more information about appearance and extension of pharmacologic drugs in surface water (2013/39/EU).

In most European regions, governments recognize that this issue was under-represented. Independent monitoring programs have been set up for a number of the active compounds, unfortunately, the programs and compounds monitored vary by region based on governmental jurisdiction.

This has created an interesting challenge for analytical scientists, who have been required to set up methods that require flexibility.

Screening Methods

HPLC and GC screening methods are widely used to gain data about a high number of compounds per sample, many with differing chemistries. These methods are not mainly optimized for the separations, but rather for speed, and method development is frequently connected to a specific equipment. The heart of most screening methods are mass spectrometric systems, these systems are limited by the speed at which they can collect and process data. In addition, databases may be required with specific information about retention times, retention time indices, fragmentation patterns, or other parameters of interest.

For screening methods, the separation of specific compounds is not as important as keeping the number of compounds in a given timeframe small enough for the mass spectrometric algorithms to give not less than 10 points per peak.

The duty of a chromatographic column in this situation is to distribute compounds with different polarity over the course of the analysis, making complete data acquisition possible for the mass spectrometer. The other requirement for the column in this instance is to give stable retention times both throughout the run and over time, as changing retention times lead to needless software adjustment. Injection to injection and batch to batch repeatability are a must.

HPLC/MS Screening Method for the determination of 66 Pharmacologically active substances

A screening method for 66+ pharmacologically active substances in waste water was developed at the Institut für Energie- und Umwelttechnik e.V. (IUTA - Institute of Energy and Environmental Technology e.V.) in Duisburg, Germany. This method is widely used for monitoring these compounds and for the conceptual design of waste water treatment plants. This method deals with a heterogeneous set of substances and the measurement requires low pH values, which may negatively affect column stability and lifetime. To validate the robustness of the method, several columns were tested. The RESTEK Raptor ARC-18 was shown to solve all of the discussed challenges.

Method parameters:

Instrument:

Pump A:            Shimadzu LC20AD Pump B:            Shimadzu LC20AD Autosampler:     Shimadzu SIL20AC Column heater:   Shimadzu CTO20AC

Mobile Phases

Channel A:     Water / 0.1% formic acid Channel B:    Acetonitrile / 0.1% formic acid

Gradient

time (min)	%B (%)
0	5
2	5
6	30
6,2	30
10	95
11,2	95
11,5	5
16	5

 

Detection System

Mass Spectrometer:     AB Sciex Q TRAP 3200 Modus:                       Scheduled MRM (sMRM)

Column oven temperature:   35°C

Injection Volume:                  20 µl

Column:     Raptor ARC-18, 50 mm x 2.1 mm; 2.7 µm

Reference Standard:   all compounds 500 ng/ml

Compound list (66 total for this run):

5-Methyl-Benzotriazol	Diclofenac	Paclitaxel
Ac-Sulfadiazin	Dimethyl_Benzotriazol	Paracetamol
Ac-Sulfamerazin	Docetaxel	Phenazon
Ac-Sulfamethoxazol	Erythromycin	Piperacillin
Allopurinol	Fenofibrinsäure	Prednisolon
Ampicillin	Flucloxacillin	Propranolol
Atenolol	Flutamid	Ritalinsäure
Azithromycin	Gemfibrozil	Roxithromycin
Benzotriazol	Ibuprofen	Roxithromycin_V
Bezafibrat	Ifosfamid	Simvastin
Bisoprolol	Isoproturon	Sotalol
Capecitabin	Ketoprofen	Sulfadiazin
Carbamazepin	Linuron	Sulfamethazin
Cefixim	Melperon	Sulfamethoxazol
Cilastin	Metoprolol	Sulfapyridin
Ciprofloxacin	Metronidazol	Tamoxifen
Citalopram	MTX	Terbutryn
Clarithromycin	Naproxen	Tetracyclin
Clenbuterol	Norfloxacin	Tramadol
Clindamycin	Ofloxacin	Trimethoprim
Cyclophosphamid	Olanzapin	Vancomycin
Diazinon	Oxacillin	Venlafaxin

 

Results

As shown in chromatogram 1, the RESTEK Raptor ARC-18 column showed peak distribution over the complete chromatogram. When used in combination with the AB-Sciex Q TRAP 3200, the method separated and quantified 77 pharmacologically active compounds in only 10 minutes.

Restek Corporation would like to thank Terence Hetzel, Dr. Thorsten Teutenberg and Dr. Jochen Tuerk from IUTA for testing our columns for robustness and suitability with their improved and complex analytical methods. IUTA/RESTEK cooperation was part of the RESTEK Academic Support Program (RASP).

Institut für Energie- und Umwelttechnik e.V. (IUTA - Institute of Energy and Environmental Technology) Bliersheimer Str. 58 - 60 D-47229 Duisburg Germany

The author would also like to thank Ty Kahler from the Restek LC-Group for reviewing this article.