CLINICAL/FORENSICS ARTICLE

Simplify and Speed Up Opiates Analysis

Using LC/MS/MS & an Allure® PFP Propyl HPLC Column

• 7-minute analysis time, for increased sample throughput.
• Faster sample prep—no derivatization required.
• Separate compounds with similar mass spectra.

Introduction

Opiates are one of the primary drug classes tested in clinical and forensic laboratories. The most common confirmation methods typically use GC/MS. These methods require derivatization of the target compounds which lengthens sample preparation time. Here we present an alternative confirmation method, by LC/MS/MS, which can be used to increase sample throughput by eliminating derivatization and shortening analysis time. Confirmation and quantification of compounds that have similar mass spectra is also possible with this procedure. Here, we use an Allure® PFP Propyl column to chromatographically separate opiates that share product ions, allowing positive identification based on retention time.

Procedure

An LC/MS/MS method was developed for the analysis of opiates. The goals of the project were to obtain baseline resolution of compounds having similar mass spectra while providing an analysis time of less than 10 minutes. To accomplish this, mass spectrometer conditions, column selection, mobile phase, and gradient profiling were evaluated and optimized.

A Shimadzu LC20 Prominence Series HPLC system coupled with an Applied Biosystems/MDS Sciex API 3200™ MS/MS system was used in this investigation. After mass spectrometry conditions were optimized for each compound, the resulting mass spectra were used to generate +MRM (multiple reaction monitoring) methods. Since MS/MS was used, we were able to target two +MRM transitions per compound to verify the identity of each compound. Table I shows the +MRM transitions and the mass spectrometer conditions. Standards contained morphine, hydromorphone, oxymorphone, codeine, hydrocodone, oxycodone, and 6-monoacetylmorphine (6-MAM) in methanol. The on-column concentration used for column evaluations was 250ng for all compounds.

Several different stationary phases were evaluated including an aqueous C18, a biphenyl, a propyl cyano, and a pentaflurophenyl propyl stationary phase. Consistent column dimensions and base silica (50mm, 2.1mmID, 5µm particle size, and 60Å pore size) were used for all phases; mobile phase conditions were optimized for each stationary phase. Mobile phases tested included: 0.1% formic acid in water, 0.1% formic acid in acetonitrile, and 0.1% formic acid in methanol in various combinations. A variety of gradient profiles also were evaluated.

Results

Although two +MRM transitions were targeted for each compound, some compounds such as codeine and hydrocodone, shared all monitored product ions (Figure 1). Since these compounds have similar mass spectra, two peaks appear in the extracted ion chromatograms. This made it necessary to separate codeine and hydrocodone chromatographically and identify compound peaks by retention time. Morphine and hydromorphine present the same challenge. The results of this study showed that of the stationary phases tested, the pentafluorophenyl propyl phase (Allure® PFP Propyl column) produced the best chromatographic separation and peak shape. Baseline resolution of opiates that shared the same product ions was achieved on an Allure® PFP Propyl column in a total analysis time of 7 minutes (Figure 2). Mobile phase gradient and composition had a significant effect on peak shape and resolution (data not shown) and optimized analytical conditions were used.

Conclusions

Here we demonstrate the Allure® PFP Propyl column, coupled with an Applied Biosystems/MDS Sciex API 3200™ MS/MS system and a Shimadzu LC20 Prominence Series HPLC, produced positive identification of opiates while reducing sample preparation time and keeping analysis time short. Use of the Allure® PFP Propyl column and the LC/MS/MS method shown here can increase sample throughput and is recommended for routine opiates analysis.

Figure 1 Codeine and hydrocodone share product ions and must be separated chromatographically.
A. Codeine LC_PH0457
B. Hydrocodone LC_PH0458 Sample: opiates Inj.: 10µL Conc: 25ug/mL Solvent: methanol Column: Allure® PFP Propyl Cat.#: 9169552 Dimensions: 50mm x 2.1mm Particle Size: 5µm Pore Size: 60 Å Conditions   Mobile Phase: A: 0.1% formic acid in water B: 0.1% formic acid in 80:20, methanol:acetonitrile Time: %B 0.0 10 3.00 50 6.00 50 6.10 10 8.10 Stop pumps Flow rate: 0.40mL/min. Ion Source: Electrospray, positive Temp: 30°C Det: Applied Biosystems/MDS Sciex API 3200™ MS/MS system IonSpray Voltage: 5500 Gas 1 65psi (448kPa) Gas 2 45psi (310kPa) Source Temp: 600°C

Figure 2 Fully resolve opiates with shared product ions (morphine/hydromorphine and codeine/hydrocodeine) on an Allure® PFP Propyl column.
Peak List:   Compound Mass 1. morphine 286* 2. oxymorphone 302 3. hydromorphone 286* 4. codeine 300** 5. 6-MAM 328 6. oxycodone 316 7. hydrocodone 300** A. Multiple reaction monitoring (+MRM) using transitions in Table 1 LC_PH0454 B. Total Ion Chromatogram LC_PH0459
See Figure 1 for conditions.

Table I +MRM Transitions for Opiates.
Mass Spectrometer Experiments: Compound Q1 Q3 Declustering Potential (V) Collision Energy (V) Morphine 286 152 46 79 Morphine 286 165 46 51 Hydromorphone 286 185 46 41 Hydromorphone 286 157 46 55 Oxymorphone 302 227 36 37 Oxymorphone 302 198 36 55 Codeine 300 152 46 85 Codeine 300 115 46 89 Hydrocodone 300 199 46 39 Hydrocodone 300 128 46 39 Oxycodone 316 240 31 39 Oxycodone 316 256 31 33 6-Monoacetylmorphine 328 211 51 55 6-Monoacetylmorphine 328 193 51 35

RESTEK PRODUCTS

HPLC Columns

• Allure® PFP Propyl 3µm & 5µm Columns (USP L43)

Guard Columns

• Allure® Guard Cartridges

ALSO OF INTEREST

• Why Derivatize?
• Analyze and Confirm Cannabinoids by LC/MS/MS
• Reliably Confirm Cannabinoids by GC/MS
• Rapid Analysis of Steroid Hormones by GC/MS

TECHNICAL ARTICLES

• View all articles