A Critical Review of Cocaine and Its Principal Metabolites Wastewater Analysis

✅ Paper Type: Free Essay	✅ Subject: Chemistry
✅ Wordcount: 5177 words	✅ Published: 8th Feb 2020

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Introduction

Illicit drug consumption monitoring has been performed in multiple direct methods such as population surveys, crime statistics, and consumer interviews. These methods are not completely reliable forms of information: they are unable of reaching most of the population, and the information collected cannot be verified (EMCDDA, 2002). An indirect method of drug monitoring has been developed from wastewater analysis. An indirect method for drug monitoring has been developed form the wastewater analysis. The quantity of illicit drugs and their metabolites found in a sewer has been used to quantify the drug consumption in its service area.

Cocaine is one of the most investigated illicit drugs, it is semi-synthetic and it derivates from the leaves of Erythroxylon coca and Erythroxylon novogranatense. It has two principal forms: freebase (cocaine sulphate) which is not water-soluble, and it is usually smoked, and cocaine hydrochloride, which is a water-soluble powder usually snorted or injected (Verebey and Gold, 1988).

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Most of the cocaine is hydrolysed before being expelled from the body. The plasma enzyme butyrylcholinesterase (BChE) or the hCE-2 liver carboxylesterase enzyme catalyse the cocaine benzoyl ester hydrolysis generating ecgonine methyl ester (EME), while another liver carboxylesterase hCE-1 catalyse the cocaine methyl ester hydrolysis generating benzoylecgonine (BE) (Zheng et al., 2008).

The cocaine type influences the metabolism pathway when assumed in the hydrochloride form it is mostly metabolized as BE while in if taken in the freebase form its principal metabolite is EME. Therefore, it is possible to monitor the cocaine freebase increase in consumption, which according to the European drug report is becoming more easily available and it is a source of concern (EMCDDA, 2018).

(Gheorghe et al.,2007)

This study will be concentrated on the need for common methodology in order to obtain comparable results for the wastewater analysis of cocaine and its metabolites. The following researches will be analysed since their focus verge on cocaine and its metabolites analysis: Gheorghe et al. (2007) Van Nuijs et al. (2009) and Castiglioni et al. (2011).

Sampling

One of the less studied and detailed reported procedures is sampling, by equalizing this procedure most researches could more easily compare their data. Rainfalls largely increase the wastewater flow, therefore, the drug amount detected during raining days will be substantially lower in respect to dry days (Ort et al. 2010).

Drug consumption varies between different days and hours especially during the weekend as reported by Reid et al. (2011) and showed in the following graphs.

Reid et al (2011)

If the sampling took place in particular days or hours, these should be reported, such as the sampling frequency. Mathieu et al. (2011) calculated that the drug quantification uncertainty range is 0.8% to 3% when the sampling frequency is 1 sample in 20 min. This is due to the variability in time and composition of toilet flushes which contain most of the substances of interest.

research	sampling
Van Nuijs et al. (2009)	Two 24-h flow dependent composite samples were taken, one on Sunday and one on Wednesday to evaluate differences in cocaine abuse during the week, no rainfalls reported.
Castiglioni et al. (2011)	24-h composite wastewater samples collected every day for 7 days with no rainfalls. sampling frequency reported in 2 of the 7 areas: one sample every 20 min, and one every 15 min.
Gheorghe et al. (2007)	24-h composite samples when possible collected before and after the weekend

Storage

Cocaine degradation is highly influenced by the wastewater matrix, which differs in every study, therefore it must be independently calculated (McCall et al., 2016). To avoid cocaine degradation, it should be acidified (pH<4) and stored at low temperature to inhibit its hydrolysis (Das Gupta, 1982). Gheorghe et al. (2007), and Van Nuijs et al. (2009) studies adjusted the wastewater pH to 2 and stored at -20 C. Castiglioni et al (2011) did not correct the pH and stored the samples at -20 C after conducting a stability study.

Analysis of drug stability after spiking (T0), after a freeze-and-thaw cycle (-20 C) (F/T), after 4 C storage for 1 day (T1) and 3 days (T3).

Extraction

To ensure that the quantity of cocaine and its metabolites found in every research are comparable the recovery percentage should aim to be high and as similar as possible.

The preferable method for drug extraction in wastewater is the solid phase extraction (SPE). In solid phase extraction, wastewater solution passes through a sorbent which traps the chemicals of interest and some impurities. The most solution is diluted the more time is needed for the sorbent porous surface to be saturated. Therefore, SPE is the most suitable extraction method since drugs in wastewater are usually present in low concentrations (ng/L). The chemicals of interest trapped in the sorbet are eluted and dried before being analysed (Bell, 2012).

The extraction is successful when all the target drug present in wastewater are transferred to the sorbent. Therefore, the interactions of the target compounds with the sorbent must be stronger than the ones with wastewater.

SPE extraction

research	analytes	Wastewater adjusted pH	sorbent	Recovery %
Castiglioni et al. (2011)	EME	2	Oasis MCX cartridge (60 mg) conditioned with 12 mL methanol and 6 mL Milli-Q water	EME 101±8
Castiglioni et al. (2006)	COC, BE	2	Oasis MCX cartridges conditioned by 6 mL of methanol, 3 mL of Milli-Q water, and 3 mL of water acidified to pH 2	COC 96±5 BE 107±9
Gheorghe et al. (2007) and Van Nuijs et al. (2009) *	COC, BE, EME	6	Oasis HLB® (500 mg) conditioned with 3 mL of a 5% MeOH in Milli-Q water	COC 95.7±5.5 BE 91.8±2.2 EME 72.5±5.3
Gheorghe et al. (2007)	COC, BE	6	Oasis HLB® (500 mg) conditioned with 5 mL of hexane, 5 mL of ethyl acetate, 10 mL of MeOH and 1 mL of Milli-Q water	COC 99.8±8.0 BE 102.3±6.6

*Van Nuijs et al., (2009) perform the SPE following Gheorghe et al., (2007) protocol and did not calculate the recovery%, assuming that it would not change.

The optimal pH, to decrease the strength of the interactions between the target drugs and the wastewater, depends on the chemical properties of the target drugs. According to the above-reported table, EME recovery improves with a lower pH. However, different sorbents have been utilized, therefore the EME optimal pH should be studied separately. The retention percentages of BE and COC are very similar in most of the studied researches this could be due to the fact that they both present the same octanol/water partition coefficient (log P) of 2.3 (Baker and Kasprzyk-Hordern, 2011).

(González-Mariño et al., 2010)

A good recovery percentage is necessary for chromatography analysis if the quantity of a compound is near the instrument limits of quantification the measurement accuracy decreases. Regarding the EME extraction, Castiglioni et al (2011) utilized a different sorbent in respect to the BE and COC one, archiving better recovery percentages: to avoid EME underrating its extraction condition should be studied separately.

During SPE, as mentioned before, some impurities will be trapped in the sorbents their complete removal could cause the loss of some of the target compounds. Therefore, some of them might be found in the samples and interfere with chromatography analysis. According to Bujak et al. (2016) molecularly imprinted solvent (MIPs), which are sorbent created for target molecules. For example, MIPs can retain BE and COC which are very similar molecules, but they cannot retain EME, such as they will not be able to trap most of the impurities. MIPs have not been tested in any of the analysed researches.

Chromatography

The extracted compounds are divided into their components that will be analysed and quantified. Liquid chromatography separation technique has been used in all the analysed papers.

Liquid chromatography is composed of a column filled with a porous compound (stationary phase), and a mobile phase: a solvent or a mixture of solvents, which flow through the column. A liquid sample is injected into the column and separated in its components depending on their affinity for the mobile and stationary phase.

Three different types of liquid chromatography have been investigated for cocaine and its metabolites separation:

Normal phase liquid chromatography (LC)	mobile phase is less polar than the stationary phase	suitable for relatively polar compounds
Hydrophilic interaction liquid chromatography (HILIC)	mobile phase is less polar than the stationary phase	suitable for hydrophilic very polar compounds
Reverse phase liquid chromatography (RP-LC)	mobile phase is more polar than the stationary phase	suitable for non-polar hydrophobic compounds

To elute two similar compounds such as COC and BE the mobile phase must be changed slightly to avoid that both compounds will be eluted at the same time preventing the separation.

Every compound will take a specific amount of time to pass through the column (retention time) and reach the detector. For every detected compound a different peak will be present on the chromatogram. (Chromacademy.com, 2018)

A compound could be identified by its retention time however it will be necessary to be analysed by the mass spectrometer to have further information about its chemical composition. Through mass spectrometry, a compound is firstly divided in its component ions, which are accelerated by a magnetic field and then detected. The bigger ions with a small charge will be the slowest to reach the detector while the smallest with a bigger charge will be the fastest this type of spectrometer is called Time of Flight. TOF-MS is utilized to detect and identify multiple compounds while triple quadrupole mass spectrometry is mostly used for specific compounds to detect. By changing the quadrupoles arrangement is possible to detect exclusively the ions target compounds (Lucini et al., 2015).

The limits of quantification of an analyte can be calculated by decreasing little by little the analyte quantity every x number of analysis, until the peak standard deviation will exceed a determinate range, such as 10%. The last quantity analysed will correspond to the limit of quantification for that analyte (Shrivastava and Gupta, 2011).

Results

research	Analytes	Chromatography	column	flow rate (μL/min)	Mobile phase	Retention time (min)	LOQ (ng/L)
Huerta-Fontela, Galceran and Ventura (2007)	COC, BE + various substances	UPLC– QqQ MS/MS	AQUITY UPLC BEH C18 (1.7 μm; 1 mm × 150 mm)	flow 500μL/min	solvent (A) acetonitrile with 0.1% formic acid; solvent (B) 30 mM formic acid/ammonium formate (pH 3.5). Gradient programme: 0-0.1 min, 5% A; 0.1-3.0 min, 5-30% A; 3.0-5.0 min, 30-80% A; 5.0-5.5 min return to initial conditions; 5.5-6.5 min, equilibration of the column	Not reported	COC 0.2 BE 0.2
Hummel et al., (2006)	BE + opioids, tranquillizers, the antiepileptics plus metabolites	RP-HPLC-ESI(+)-QqQ MS/MS	Synergi Polar-RP 80 Å (150 × 3 mm, 4 μm)	Flow 500μL/min	Mobile phase: acetonitrile (A) and 10 mM ammonium formate in water, adjusted to pH 4 with formic acid (B). Gradient programme: 0-5 min- 10% A, linearly increased to 80% A within 13 min, return to the initial conditions 10% A within 2 min which were kept for the last 10 min	BE 11.3	BE 10
Gheorghe et al., (2007)	COC, BE EME	HILIC- MS/MS	ZORBAX Rx-SIL (2.1mm×150mm × 5μm) (Agilent) + guard column (2.1 mm×12.5 mm×5 μm) containing the same stationary phase as the HILIC column	flow 250 μL/min	solvent A: ammonium acetate 2 mM/acetic acid buffer (pH=4.5); solvent B: ACN. Gradient programme: 0–1 min 80% B; 1–10 min 80% to 40%B; 10–18 min constant 40% B; 21–31 min: column equilibration with 80%B.	COC 9.3 BE 4.7 EME 13.6	COC 1 BE 0.5 EME 20
	COC, BE	RP-HPLC MS/MS	C18 reversed-phase column, Zorbax Extended C18 (2.1 mm×50 mm×3.5, + guard column (2.1 mm×12.5 mm×5 μm) containing the same stationary phase	flow 250 μL/min	solvent A: water/AcN 98:2, 10 mM formate buffer, pH=3; solvent B: AcN Gradient programme: starting eluent 97% solvent A and 3% solvent B; 0–6 min 3% to 40% solvent B; 6–12 min 40% to 90% solvent B; 12– 22 min equilibration of the column with the starting eluent.	COC 7.6 BE 6.4	COC 4 BE 2
Castiglioni et al., (2011)	EME and other cocaine metabolites	HILIC-LC-MS/MS	X-Bridge HILIC	flow 300 μL/min	ammonium formate 5 mM in water acidified to pH 4 with formic acid as solvent A, and acetonitrile as solvent B. Gradient programme: starting with 5% of eluent A and 95% of eluent B, followed by a 12-min linear gradient to 50% of eluent A, a 3-min isocratic elution and a 1-min linear gradient to 5% of eluent A maintained for 7 min.	Not reported	EME 2.6
Castiglioni et al., (2006)	COC, BE, + norcocaine, cocaethylene	RP-HPLC-ESI(+/−)-QqQ MS/MS	XTerra MS C18, 100 × 2.1 mm, 3.5 μm	flow, 200 μL/min	solvent A, acetic acid 0.05% in water; solvent B, acetonitrile. Gradient programme: 0 to 30% of B in 18 min, to 100% B in 2 min (2-min hold) and to 0% B in 2 min (8-min hold)	COC 14.9 BE 14.3	COC 1.4 BE 1.98
Van Nuijs et al. (2009) *	COC, BE, EME	1)HPLC ESI MS 2) UPLC MS/MS	1)a ZORBAX Rx-SIL HILIC (2.1 mm x 150 mm, 5 mm) (Agilent) and a guard column with the same stationary phase 2)C18 UPLC	Not reported	See HILIC Gheorghe et al., (2007)	Not reported	See HILIC Gheorghe et al., (2007

Van Nuijs et al. (2009) * analysis executed by two different laboratories, the same procedure and LOQ of Gheorghe et al., (2007) during HILIC analysis have been reported, the LOQ could have changed due to the different wastewater matrix but they have not been reported.

According to the results in the table HILIC columns provides the better separation between COC and BE in respect to the other analysed columns. EME is only been investigated by HILIC LC due to its high hydrophilicity, however very different results have been archived: Castiglioni et al. (2011) EME LOQ 2.6 (ng/L), Gheorghe et al. (2007) EME LOQ 20 ng/L). The principal changes in these studies could be due to the different solvent and gradient programmes utilized since both studies adopted a HILIC column as stationary phase. Gheaorghe et al. (2007) reported good results for BE and COC LOQ, therefore, their analysis should be unchanged and divided from the EME one, which should be performed with the mobile phase described by Castiglioni et al. (2011).

Hummel et al. present the highest BE LOQ this is probably due to the buffer choice since the results of Huerta-Fontela, Galceran and Ventura, (2007) research suggest that increasing the amount of the ammonium formate buffer and slightly decreasing the pH cleaner chromatogram peaks can be observed as displayed below. These adjustments seem to be valid also for the reverse phase chromatography since Gheorghe et al. (2007) reported better LOQ results utilizing the same buffer amount as Hummel at al. with a lower pH.

(a) ammonium acetate buffer (10 mM, pH 4.0); (b) ammonium formate buffer (10 mM, pH 3.5); (c) ammonium formate buffer (20 mM, pH 3.5); (d) ammonium formate buffer (30 mM, pH 3.5)

With a gradient programme: 0-3.0 min, 5-20% ACN; 3.0-5.5 min, 20-60% ACN. COC is n.12 and BE is n.9.

Triple quadrupole is the most utilized between the analysed papers probably for its selectivity and its quantification ability.

Backward calculations

Van Nuijs et al. (2009) base their backward calculation on the formula described by Zuccato et al. (2005) the cocaine amounts have been calculated by multiplying the BE g/day for a factor of 2.33 which takes in account the COC percentage execrated as BE (45%) and the BE/COC molar mass ratio. The changes in BE excretion due to the use of freebees cocaine have not been considered such as the sampling uncertainty the increase of BE due to cocaine hydrolysis in wastewater and the recovery percentage. The formula utilized by Baker, Barron and Kasprzyk-Hordern (2014) has partially addressed the above-mentioned problems, however, if the drug quantities have been calculated with different formulas their comparison accuracy will decrease.

mg/day 1000/people

Load – quantity of drug target residue (DTR) arriving at the WWTP (g/day);

Excretion – quantity of DTR execrated arriving at the WWTP

MWPar – molecular weight of the parent compound

MWDTR – the molecular weight of the DTR.

OS – quantity of DTR present in wastewater due to other sources other than the parent compound (mg/day 1000/people).

Conclusion

Wastewater analysis is a good method for cocaine estimation in wastewater, however, the calculations of cocaine consumption are not as reliable because of the high uncertainty of the percentage cocaine and its metabolites execration, the different sampling methods, the stability of the drugs in wastewater, the different recovery percentage and the backward calculation formulas utilized.

This study observed that performing cocaine and its metabolites solid phase extraction and chromatography analysis when EME has been separated from the BE and COC, the EME recovery % and LOQ increased significantly. The HILIC-LC-tandem MS has provided the best separation of cocaine and its metabolites between the analysed techniques.

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