Carl Sanchez

Chronic pain affects approximately 86 million people in the United States (1). Pain management centers have been established all over the country to help treat these chronic conditions. During treatment, routine testing is critical to monitor compliance and prevent abuse. Because the average age of healthcare patients continues to increase, the demand for pain management is growing rapidly. To keep up with demand, less expensive, faster, more accurate, and reproducible methods for analyzing the presence and concentration of pain management medications in vivo are needed. 

Urine is commonly used for pain management testing as it is a copiously available bodily fluid that can be collected easily. The constituents in urine and their concentration vary significantly between patients and are dependent upon diet, hydration, and health condition. Variations in the endogenous components in urine can impact quantitation in methods using liquid chromatography with mass spectrometric detection (LC–MS-MS). Coelution of endogenous urine components with the analytes of interest can cause matrix effects that result in enhancement or suppression of the MS signal, which impacts the quantitative accuracy of the assay. Removal of endogenous components using solid-phase extraction (SPE) is an effective method for reducing potential matrix interferences before LC–MS-MS analysis.  

Typically SPE and gas chromatography (GC)–MS have been used to perform the analysis of opioids in urine. Most GC–MS methods require 7 min or longer from injection to injection. The method described here couples sample cleanup and concentration using strong-cation-exchange SPE with rapid LC–MS-MS analysis (<2 min) for 13 commonly prescribed pain management compounds in human urine.  

A bioanalytical validation for a new SPE and LC–MS-MS procedure for the analysis of pain management drugs in human urine was performed. Five different lots of urine samples were spiked at therapeutically relevant concentrations with 13 commonly prescribed drugs, including hydrocodone, naltrexone, tramadol, and fentanyl. All drug standards were obtained from Cerilliant Corporation (Round Rock, Texas). The lower limit of quantitation (LLOQ) was targeted at 10× below the therapeutic level with less than 20% RSD at LLOQ. The upper limit of quantitation (ULOQ) was set at 10× above the therapeutic cutoff. 

Urine samples were first hydrolyzed to convert the glucuronide metabolites to native form using β-glucuronidase. This was done by first weighing 1 g of β-glucuronidase and diluting with 20 mL of deionized water. A 100-µL aliquot of this solution was added to 1 mL of urine and vortexed to mix (6200 units in 1 mL of urine). Samples were incubated for 2 h at 60 °C and then were centrifuged for 10–15 min at 9500–13,000 rpm to form a pellet of protein and solids. The clarified supernatant (~1 mL) was then removed and diluted with 1 mL of 100 mM ammonium acetate buffer (pH 5.95).  

Cleanup was performed using polymer-based strong-cation-exchange media (Strata-X-C 30 mg/3 mL, Phenomenex, Torrance, California) to remove endogenous matrix components. Analysis was performed using a high performance liquid chromatography  (HPLC) system (1200SL, Agilent, Santa Clara, California) coupled to a 4000 QTRAP MS system with a Turbo V ion source (Applied Biosystems, Foster City, California). The chromatographic column used was a 50 mm × 2.1 mm, 2.6-µm C18 (Kinetex, Phenomenex, Torrance, California), which is based upon new core-shell particle technology.  

Equilibrate: 1 mL 100 mM ammonium acetate buffer (pH 5.95) 

Load: Pretreated sample diluted with 1 mL 100 mM ammonium acetate buffer (pH 5.95) 

Wash: 2 × 1 mL 100 mM ammonium acetate buffer (pH 5.95) 

Dry: Full vacuum for 2–5 min to remove residual water and methanol 

Elute: 2 × 0.5 mL 60:24:16 ethyl acetate–isopropanol–ammonium hydroxide 

Evaporate to dryness using nitrogen gas and a heating block at 45 °C, then reconstitute in 1 mL of initial mobile phase for LC–MS-MS analysis. 

The analysis of all 13 pain management drugs was completed in less than 1.5 min with a total gradient cycle time from injection to injection of less than 2.2 min (Figure 1). The use of the core-shell HPLC column provided extremely narrow chromatographic bands with average peak widths at base of ~1 s. These extremely narrow peak widths challenged the scan rate of the MS system operated in triple-quadrupole mode and required the use of scheduled MRM transitions to ensure enough scans per peak to maintain chromatographic resolution. The narrow peak widths also translated into significant increases in MS sensitivity, requiring the dilution of some samples by 20-fold to bring concentrations within the dynamic range of the system. 

The columns used provided efficiencies similar to sub-2 µm particles at back pressures that allow them to be used on conventional HPLC systems. Figure 2 shows the core-shell particle geometry. These particles are manufactured by growing a 0.35-µm porous layer on a 1.9-µm nonporous core. The result is 2.6-µm particles.. 

Table I summarizes the results obtained using the procedure described. The chromatographic flow rate of 1.2 mL/min enabled the fast separation of the 13 compounds and the internal standards, with retention times ranging from 0.22 min (2 #3215; void, 0.075 min) for morphine to 1.2 min for methadone. Some internal standards were used as reference for analytes where the deuterated analog was not readily available. The analytical method gave an LLOQ of 2.5 ng/mL with accuracy of 83–98% for all compounds with the exception of fentanyl, which had an LLOQ of 0.05 ng/mL due to its low therapeutic concentration. At LLOQ and 3 × LLOQ, most RSDs were well below 15%, indicating a wide linear working range for all compounds. Method performance at cutoff was better than at the 3 × LLOQ, indicating a possible dilution error during sample preparation. Future work will be done to confirm this. The cutoff concentrations are defined as 300 ppb for all compounds except for fentanyl. Due to the relatively high pharmacological efficacy of fentanyl, ~50 times greater than morphine, the linear analytical range of fentanyl was 50× lower than the other opioids. At cutoff concentrations, all compounds were analyzed with 84–102% accuracy and 5% or less RSD values. Matrix effects were evaluated by determining lot-to-lot precision and accuracy using five different urine donors. At the cutoff concentration the RSD values were less than 7% with high accuracy across all donors. Figure 3 shows an example calibration graph spanning the linear range of 2.5–2500 ppb for morphine, with 1/

Table I: LCâMS-MS results from bioanalytical validation protocol 			

The high selectivity of LC–MS-MS greatly reduces interferences that affect analyte quantitation. This high selectivity can reduce sample cleanup requirements; however, reduced sample cleanup allows more endogenous material to accumulate in the MS source, which ultimately can increase the frequency of maintenance. The use of SPE for sample clean-up greatly reduces matrix interferences while also significantly reducing the contaminant load on the MS source. The effect of SPE was most dramatic for fentanyl due to its 5 ng/mL cutoff concentration. Using a 30-mg bed mass significantly reduces the solvent volume required for the conditioning, wash, and elution steps. This reduced solvent volume translated into significant cost and time savings for the subsequent blowdown steps.  

The SPE protocol utilized has been optimized for the 13 analytes and internal standards. The basic p

 values of the cationic analytes are all close to 8. The buffer chosen, ammonium acetate, is adjusted to pH 5.95 in order to protonate and charge the cationic analytes at 2 pH units below their p

 values. This pH ensures the target analytes are fully protonated maximizing retention and recovery with the sulfonic acid ligand on the SPE medium. Use of ion-exchange retention allows 100% methanol wash to more effectively remove endogenous contaminants including those with lower basic p

 values, such as urobilin, which, when present, may appear yellow. The selectivity of the elution solvent composition, 60:24:16 ethyl acetate–isopropanol–ammonium hydroxide, was optimized to allow elution of the analytes of interest while leaving any remaining endogenous components on the sorbent. Optimized wash and elution solutions yielded clearer and cleaner extracts (Figure 4) and more reproducible and accurate chromatography. The sorbent mass was also optimized to 30 mg of media in a 3 mL tube. The high ion-exchange capacity of the media allows processing small sample volumes even at low concentrations. Due to the high sensitivity of the method, only 1 mL of urine was required for SPE. Using less sorbent reduces cost and solvent consumption and reduces elution volumes, thereby saving time during evaporation, an important consideration for high sample throughput and increased productivity. 

Ultrahigh efficiency and fast chromatographic separations result in very narrow peak widths, enhancing resolution and sensitivity. However, these very narrow peaks also present detection challenges when using MS. This is especially true for this method, in which two MRM transitions must be monitored concurrently for each of the thirteen compounds and one for each of the eight internal standards. To optimize the acquisition rate on the MS system, a scheduled MRM window of 6 s was used to get ~20 data points per second (15–18 Hz experimentally). Sufficient sampling of the chromatographic peak is important to maintain resolution between closely eluted bands including other analytes as well as possible interferences. The MRM settings used here are highly recommended because the separation provided by the column produces very narrow, low-volume peaks. 

Additionally, when using the columns described here, LC–MS-MS instrumentation should be optimized so the narrow analyte bands are not compromised by extracolumn band broadening. The instrumentation used for this experiment was optimized by bypassing the MS grounding union, eliminating the diverter valve, and utilizing a near-zero dead volume in-line column filter (KrudKatcher Ultra, Phenomenex) to protect the column. It is recommended that the narrowest internal diameter and shortest lengths of connecting tubing possible be used. The effects of dispersion also can be minimized by using high flow rates, injecting the sample in weaker solvent than the initial mobile phase, and running steep gradients when possible.  

A bioanalytical method validation was performed for 13 commonly prescribed pain-management drugs in human urine. Accuracy, precision, and lot-to-lot matrix effects were all within expectations for bioanalytical mthods. The use of the polymeric SPE discussed here greatly reduces matrix interferences while also providing clean sample extracts. The 30 mg/3 mL format reduced the amount of solvent used for processing and the time required for blowdown steps. The columns used provided fast run times and improved method sensitivity. This prevalidated solution greatly reduces the time required for implementation in any laboratory interested in analyzing pain management samples. 

13 pain management opioids were resolved in 1.5 min with 1-s wide peaks at base on a 50 mm × 2.1 mm, 2.6-µm 

 C18 column. Sensitivity, reproducibility, and productivity are dramatically improved after efficient sample cleanup and concentration using the SPE described here. The combined SPE LC–MS-MS analysis provides significant reductions in analysis time, labor requirements, and cost per sample while increasing sensitivity, accuracy, and precision.  

 are with Phenomenex, Inc., Torrance, California.  

(1) American Chronic Pain Association website. 

Articles

Fast turnaround time is critical in the clinical testing environment. Here, fast liquid chromatography (LC) technologies were utilized for the comprehensive assay of commonly prescribed pain management drugs in under 2 min. The use of fast LC also provided significantly improved sensitivity. A mini-validation for these analytes in human urine was performed and acceptable values for accuracy, precision, linearity, lot-to-lot variability, and matrix effects were demonstrated for each analyte.

Development of a High-Throughput LC–MS-MS Assay for 13 Commonly Prescribed Pain Management Drugs from Urine with Cleanup Using Solid-Phase Extraction

Coupling mass spectrometry (MS) to high performance liquid chromatography (HPLC) methods has become commonplace over the last two decades as many take advantage of both the sensitivity and the identity aspects of such instrumentation. The specificity of tandem MS using parent–daughter combinations (multiple reaction monitoring [MRM], for example) allows for quantifying individual compounds in a complex mixture, even when such compounds are not fully resolved from other analytes in a mixture. However, some resolution between analytes and ion-suppressing impurities is required for good MS quantitation. Combining shorter HPLC columns with MS has been the solution for developing sensitive methods for quantitating several analytes in a single analysis.  

Over the last five years, several instrument and column manufacturers have invested much in marketing and advertising about ultrahigh-pressure LC (UHPLC) and the advantages of switching to higher efficiency columns for analytical separations. Showing performance gains for HPLC applications using UV detection by switching to UHPLC columns and instruments has been a rather straightforward process. Sampling rates of newer UV detectors are such that a large number of data points per second can be acquired, allowing one to visualize and quantitate the improved chromatography that UHPLC columns deliver. With LC–MS methods, visualizing improvements in chromatography are less apparent. The scan rate for MS instrumentation can sometimes be limited to a few points per second. This rate can be even slower for methods that scan large molecular weight ranges or when numerous parent–daughter pairs are being acquired. In addition, extracolumn volume in the MS interface or detectors postcolumn can mask any separation gain from a UHPLC column. As a result, realized performance gains for LC–MS methods using UHPLC have been significantly less than standalone LC methods. 

Recently, a new type of UHPLC column has been introduced using core-shell media. These media use unique particle geometry versus the standard fully porous silica media; a nonporous core particle is surrounded by a thin porous layer of functionally bonded silica. The improved porosity and mass transfer of such media are much higher than that of fully porous media of the same particle size, resulting in performance on par with fully porous media much smaller than the core-shell media. In the case of the major core-shell products on the market, the core-shell media are slightly larger than 2.5 µm in size with performance on par with the major sub-2-µm media on the market with back pressures more in line with 3-µm fully porous HPLC media. The greater implication of such core-shell media is that one could get UHPLC performance on their existing HPLC system without having to buy a new ultrahigh pressure system.  

Efforts were undertaken to adapt various existing LC–MS methods using standard HPLC systems to using the new core-shell media. Examples of high-throughput applications for pharmaceutical and environmental analysis were developed using core shell media. Key in developing such methods include maintaining reasonable resolution of key analytes from ion suppressing impurities while balancing the limitations of the MS being used for the analysis.  

Chromatography standards (amphetamine, aflatoxin, sulfa drugs, and antibiotic mixtures), urine, and plasma were purchased from Sigma Chemical (St. Louis, Missouri). Various buffers and solvents were purchased from EMD (San Diego, California). Gemini 5 µm C18 (150 mm × 3.0 mm) and Kinetex 2.6 µm C18 and PFP (both 50 mm × 2.1 mm) HPLC columns were obtained from Phenomenex (Torrance, California). StrataX, StrataX-C, and Strata Florisil solid-phase extraction (SPE) tubes (50 mg/3 mL tubes) also were used. Food matrices were obtained from local grocery sources. Test samples were spiked into separate sample matrices. For the pharmaceutical–toxicology sample, amphetamines were spiked into urine. For environmental analysis, aflatoxin was spiked into peanut butter, sulfa drugs in honey, and an antibiotic mixture was spiked into a meat sample. While every SPE procedure was unique to the application, all had several common features: sample is diluted then loaded on their specific SPE cartridge; cartridge is washed, then analyte is eluted with an organic mixture. Samples are then dried down and reconstituted in aqueous mobile phase before injection on HPLC.  

All samples were injected on an  HP1100 HPLC system with an autosampler, solvent degasser, and multiwavelength detector (all from Agilent Technologies, Santa Clara, California). HPLC-UV data were collected using Chemstation software (Agilent Technologies). The aqueous mobile phase used was either 0.1% formic acid in water or a mixture of formic acid with ammonium formate. For organic mobile phase, either acetonitrile or methanol was used. Various gradient methods were used based upon the analyte mixture and columns used. For LC–MS analysis, the HPLC instrument was linked postcolumn (excluding the UV detector) directly to the electrospray ionization (ESI) interface of an API3000 tandem mass spectrometer (Applied Biosystems, Foster City, California). While individual MS parameters differ based upon the application, in most cases, the MS system was run in positive ion mode using MS-MS multiple reaction monitoring, in which analytes were quantitated using specific parent– daughter ion transitions. In many cases, a multiply deuterated internal standard was used for quantitation.    

The promise of UHPLC has been to shorten existing HPLC methods using 3-µm and 5-µm media up to 10-fold or more using new UHPLC systems and associated sub-2-µm media. While such dramatic increases in throughput are conceivable for methods using UV detection, for LC–MS detection, one is limited by the sampling rate of the MS detector. Depending upon the dwell time needed for sensitivity and number of transitions being monitored, one can potentially be limited to only a two- to threefold decrease in analysis time simply because the mass spectrometer might not be able to collect enough data points across an analyte peak for accurate quantitation. This has become more readily apparent with the introduction of core-shell media with performance on par with sub-2-µm columns while operating at standard HPLC back pressures. Such versatility allows for increased throughput with existing LC–MS system without having to purchase a new system that can tolerate higher backpressures. An example of this is shown in Figure 1 and Figure 2. In Figure 1, a mixture of amphetamines was run on a fully porous 5-µm C18 column (150 mm × 3.0 mm) using an 8-min run time with good resolution between individual components (the 

 internal standards obviously are eluted closely to their natural counterparts). Transferring the method to using a core-shell media column (Kinetex 2.6 µm C18, 50 mm × 2.0 mm, Figure 2), both the column and gradient time are shortened resulting in an analysis time around 3 min, even with the additional analytes in the mixture. The increased efficiency (plates/m) of the core-shell column allows for shortening column length while maintaining resolution; the van Deemter plot for 2.6-µm core-shell columns more closely resemble the plot for a 1.7-µm fully porous media, resulting in better performance at higher mobile phase linear velocities.  

A similar example of the utility of using core-shell columns to decrease the run time of an existing LC–MS method is shown in Figure 3 and Figure 4. In this case, a sulfa drug mixture is extracted from honey using SPE and injected on a fully porous 5-µm C18 column (150 mm × 3.0 mm) with a run time of 10 min (Figure 3). The gradient and column are again shortened (a 2.5-min gradient on a core-shell Kinetex 2.6 µm C18 column 50 mm × 2.1 mm) with concurrent increase in mobile phase linear velocity (Figure 4). Similar to what was shown in the previous example, the analysis time was decreased around threefold, and critical resolution between critical components is maintained without having to use a specialized UHPLC system. In addition to the platform compatibility that lower operating back pressure of core-shell media provides, one could conceivably operate at even higher linear velocities to further reduce run times. However, flow rates must be balanced with the sampling rate of the mass spectrometer. If peak widths are narrowed excessively by faster separation methods, then peak shape distortion will occur, resulting in poor method quantitation. In Figure 4, this is only slightly apparent as negative dips due to peak smoothing. In Figure 5, peak distortion is slightly more noticeable. Figure 5 is an example of an aflatoxin LC–MS method using a core-shell Kinetex 2.6 µ PFP column (50 mm × 2.1 mm). Pentafluorophenol phases are particularly popular for separation of geometrically unique polyaromatic analytes. In the case of microtoxins, PFP phases do a particularly good job at separating different aflatoxins. In this LC–MS example, all the major aflatoxins are well separated in under 2 min with good quantitation at the 50-ng level, however, very slight distortion is observed due to a slightly low sampling rate of the mass spectrometer. Such results emphasize the balance needed in developing LC–MS methods using core-shell and other high performance media: analysis speed, analyte sensitivity, and quantitation all must be factored into an LC–MS method based upon the capabilities of the mass spectrometer. The use of a PFP column for this separation also emphasizes the utility of using alternate selectivities for LC–MS applications. The use of a C18 column for this application would require a much longer analysis time because of the limited selectivity that C18 has for aflatoxins. For many applications, an alternate selectivity media can allow for shorter run times due to their greater selectivity for specific compound mixtures.  

While there can be limitations on a high-speed LC–MS method based upon the instrument platform used, Figure 6 shows an example of the tremendous power of LC–MS analysis. In this example, a mixture of 30 different antibiotics are extracted from a meat sample by SPE and run on a core-shell Kinetex 2.6 µm C18 column (50 mm × 2.1 mm) and individual MRM traces are overlaid. The 30 different analytes are partially resolved in a gradient of 8 min, and either positive or negative ion mode was used depending upon the analyte being detected. Because of the large number of analytes and the slow MS sampling rate, it is obvious that the peak shape of several analytes are distorted significantly,  resulting in compromised quantitation. In addition, coeluted analytes can potentially suppress the electrospray efficiency, resulting in further inaccuracies in quantitation. However, such a broad method can be very effective for widespread detection of analytes in which exact quantitation is not necessary.  

UHPLC media have allowed for greatly reduced run times, resulting in significant improvements in throughput for analytical methods. These improvements have been especially profound when using core-shell media, in which improved throughput and performance can be realized with existing HPLC systems without the need for dedicated ultrahigh-pressure instruments. While adapting existing HPLC methods can be fairly easy, with LC–MS methods, the specific limitations of the mass spectrometer being used must be considered. For complex MS acquisition programs, sampling rates can approach the peak widths of some analytes separated on a core-shell column. In such cases, peaks can be distorted, resulting in poor quantitation. As a result, one might not be able to completely realize the full benefit in throughput that core-shell columns provide for LC–UV methods. With a balanced view of scan rates, sensitivity requirements, and chromatographic peak widths, one can develop rapid and accurate LC–MS methods using core-shell and other UHPLC media. In addition, the different phases of core shell media (C18, PFP, or hydrophilic interaction chromatography) offer selectivity options that should be considered when developing optimal high-speed LC–MS methods.  

 are with Phenomenex, Inc., Torrance, California.

Adapting the use of "ultrahigh" performance chromatography for liquid chromatography–mass spectrometry (LC–MS) applications requires specific considerations in integrating the instrument platforms. Mobile phase options are limited to volatile buffers, and slow MS sampling rates can limit throughput advantages that such next-generation media offer. High-throughput LC–MS methods of different relevant pharmaceutical and environmental mixtures were developed using ultrahigh performance core-shell media. Such methods were developed using standard HPLC systems and back pressures, showing the ease and utility of using core-shell media for increasing throughput of LC–MS methods.

Carl Sanchez

Articles

Development of a High-Throughput LC–MS-MS Assay for 13 Commonly Prescribed Pain Management Drugs from Urine with Cleanup Using Solid-Phase Extraction

Translating HPLC Performance Gains of Core-Shell Media to LC–MS Applications