Rapid, Sensitive HPLC/TOF-MS Analysis for Cocaine Using an Allure® PFP Propyl

By Kristi Sellers, Clinical/Forensic Innovations Chemist, Julie Kowalski, Innovations Chemist, and Becky Wittrig, Ph.D., HPLC Products Marketing Manager

Introduction

When introduced into the body, cocaine produces three main metabolites: benzoylecgonine, ecgonine, and ecgonine methyl ester. To determine the presence of cocaine and/or these metabolites, urine samples are screened using enzyme immunoassay, and positive results are confirmed using gas chromatography/mass spectrometry. Mass spectrometry is widely used for confirmation because MS data provide positive identification based on mass spectral information, and can be used as confirming evidence in courts of law. Although GC/MS methods are well established, and provide excellent confirmation data, analysis time is long and sample preparation, which includes derivatization, is labor-intensive and time-consuming. Figure 1 shows a GC/MS analysis of TMS derivatives of cocaine and its metabolites, with an analysis time of 14 minutes.

 

A less commonly used, but equally valuable, chromatographic method for identifying and quantifying cocaine and its metabolites is high performance liquid chromatography coupled with mass spectrometry (HPLC/MS). The analytes do not have to be derivatized for this approach, greatly reducing sample preparation time and making HPLC/MS more attractive to the analyst. A well-chosen stationary phase and a mobile phase with a high organic content allow efficient desolvation and ionization in the MS, and permit detection of cocaine and its metabolites at low picogram levels on-column. HPLC/MS, like GC/MS, provides reproducible, reliable data that can be used in court.

The HPLC/MS method developed in this investigation provides symmetric peaks and excellent sensitivity in a reduced analysis time.

Experimental

HPLC/time-of-flight MS data were obtained using an Allure® PFP Propyl column (pentafluorophenyl propyl stationary phase) and a C18 column (octadecyl column phase). HPLC/MS/MS data also were reviewed, and were compared to the HPLC/TOF-MS data.

Reference standards of cocaine, benzoylecgonine, and ecgonine methylester were prepared at various concentrations in the mobile phase. Final on-column concentrations are listed in Table 1. Deuterated cocaine was used as the internal standard. HPLC methods were optimized for each stationary phase and were evaluated for identification and quantification of each compound (Table 2). We focused on choosing the stationary phase and conditions that provided maximum sensitivity for all compounds in an acceptable analysis time and with appropriate peak symmetry.

Cocaine, benzoylecgonine, and ecgonine methylester are hydrophilic, basic compounds with pKa values greater than 8. Consequently, buffer salts or ion-pairing agents and a mobile phase with a low organic content are needed to ensure adequate retention on a typical C18 stationary phase. Retention can be adequate under these conditions (Figure 2A), but the highly aqueous mobile phase causes poor MS response, due to inefficient desolvation, and the salts cause ion suppression during ESI. Under optimal screening conditions, limits of detection of 1ng/mL for cocaine and 5ng/mL for benzoylecgonine have been reported to be 10pg and 50pg on-column, respectively (10µL injection) for the C18 stationary phase.

In contrast, a high-organic mobile phase can be used with an Allure® PFP Propyl column (Table 2). This combination provides not only adequate retention and short analysis times, without modifiers, but also excellent sensitivity. All target compounds are eluted from the 30 x 2.1 mm column within 3 minutes, with reproducible responses (Figure 2B). S/N:RMS values greater than 90 indicate excellent sensitivity at 5.0pg on-column for all compounds; values of 16 and greater indicate adequate sensitivity for most compounds at 0.5pg. For each compound the relative standard deviation (%RSD) for intensity is less than 10% across a broad concentration range, excepting only the 0.5pg value for ecgonine methylester (Table 1).

HPLC/ESI/MS/MS also is a viable approach for detecting cocaine and its metabolites. Like HPLC/TOF-MS, it reduces sample preparation time and analysis time, and provides reliable, reproducible results at the picogram level. Figure 3 shows cocaine and ecgonine methylester detected at 1.6pg and 2.8pg, respectively. Again, the Allure® PFP Propyl pentafluorophenyl propyl stationary phase and highly organic mobile phase were needed to achieve this limit of detection. Analysis time is less than 5 minutes; peak shape is excellent.

Conclusions

HPLC/TOF-MS and HPLC/ESI/MS/MS are excellent alternate chromatographic confirmation methods for cocaine and its metabolites. Relative to GC/MS, HPLC reduces sample preparation time by eliminating derivatization and reduces analysis times from 14 minutes to less than 5 minutes. Our study demonstrated that Allure® PFP Propyl, a bonded pentafluorophenyl propyl stationary phase, in combination with a highly organic mobile phase, provides excellent sensitivity, symmetric peaks, and short analysis times.

Table 1 Mean relative standard deviations for cocaine and its metabolites are substantially less than 10% for all on-column quantities but one, by HPLC/TOF-MS (Allure® PFP Propyl column).
On-column Amount	Mean % RSD (n = 7)
COC	BZE	EME	Cd3
250ng	1.9	3.2	1.5	3.2
125ng	0.4	4.7	1.5	4.0
25ng	0.5	4.8	0.6	4.4
5ng	0.8	2.9	1.5	2.1
2.5ng	1.7	4.2	3.2	2.4
0.5ng	5.7	0.7	0.9	2.8
5pg	7.4	8.1	7.8	5.1
0.5pg	4.3	8.0	40.7	6.3
COC — cocaine; BZE — benzoylecgonine; EME — ecgonine methylester; Cd3 — cocaine-d3.

Table 2 HPLC/TOF-MS conditions for monitoring cocaine and its metabolites.
Sample:	analytes in 10µL mobile phase
Column:	Allure® PFP Propyl	C18
Cat.#:	9169532
Dimensions:	30mm x 2.1mm	30mm x 2.1mm
Particle size:	5µm	5µm
Pore size:	60Å
Mobile phase:	A - acetonitrile B - 5mM ammonium formate in water, pH=3.0
A:B:	80:20	20:80
Flow:	0.6mL/min.
Temp.:	25°C
Detection:	MS, Micromass LCT Premier
ESI:	positive
Capillary:	3000V
Sample cone:	20V
Desolvation temp.:	250°C
Source temp.:	120°C

Figure 1 GC analysis of cocaine and its major metabolites requires approximately 14 minutes.
	ecgonine methyl ester (TMS) ecgonine (TMS) cocaine coca ethylene benzoylecgonine (TMS) 30m, 0.25mm ID, 0.25µm Rtx®-5 (cat.# 10223) 1.0µL split injection of cocaine and cocaine metabolites Oven temp.: 150°C to 320°C @ 10°C/min. Inj. / det. temp.: 250°C / 300°C Carrier gas: helium Linear velocity: 30cm/sec. set @ 50°C FID sensitivity: 2.56 x 10-10 AFS Split ratio: 30:1

Figure 2 Extracted ion chromatograms of cocaine, benzoylecgonine, and ecgonine methylester, using HPLC/TOF-MS. Analysis is completed in less than 3 minutes.
Peak # ID Mass RT (min.) A) C18 column 1 EME 200 0.33 2 COC 304 0.43 3 BZE 290 0.57 B) Allure ™ PFP Propyl column 1 EME 200 1.27 2 BZE 290 2.00 3 COC 304 2.33

 

Figure 3 Extracted ion chromatograms of cocaine and ecgonine methylester, using an Allure® PFP Propyl column and HPLC/ESI/MS/MS.

Peak List: EME (ecgonine methyl ester) COC (cocaine) Sample: Inj.: 10µL Conc.: 1µg/mL Solvent: water Temp.: 4°C Column: Allure® PFP Propyl Cat. #: 9169532 Dimensions: 30 x 2.1mm Particle size: 5µm Pore size: 60Å Conditions: Mobile phase: 5mM ammonium formate, pH 3.0: acetonitrile (10:90, v/v) Flow: 0.6mL/min. Column temp.: ambient Det.: PE/Sciex API 3000 Interface: turbo ion spray, ESI Interface temp.: 150°C Ion mode: positive ESI probe voltage: 5000V Orifice: + 71V Ring: + 265V Collision gas: nitrogen Collision gas pressure: 2.2 mTorr Collision gas energy: 28 eV (COC) 26 eV (EME) Electron multiplier: 2100 volts Auxilary gas flow: 7000cc/min. Nebulizer gas setting: 15lb/in.2 Curtain gas setting: 12lb/in.2

REFERENCES

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