Michael McGinley

A method is illustrated for the simultaneous analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human urine samples. The method described uses a simple dilution of the urine sample followed by fast (<7-min) liquid chromatography–tandem mass spectrometry (LC–MS-MS) analysis with detection below 25 ng/mL. When tested with a large number of patient samples, the method was found to be rugged, reproducible, and free of interference by matrix contaminants.

Monitoring illicit drug use through laboratory testing has become a cornerstone in reducing substance abuse and focusing efforts on those stricken with addiction issues. Although many methods have been developed for detecting commonly abused drugs, monitoring individuals for alcohol consumption is hampered by the rapid metabolism of alcohol which makes abuse undetectable. Until recently this rapid metabolism has limited alcohol testing to a few hours after drinking stops. New developments have made it possible to detect alcohol abuse in the 3–5 days preceding the test by identifying the presence of alcohol metabolites. This detetection is especially important in monitoring alcohol use in settings in which alcohol use is prohibited: alcoholism and drug abuse treatment, professional assistance programs, abstinence as a condition of probation, child custody issues, or for people under the legal drinking age (1).

Beverage alcohol, also known as ethanol, is metabolized by the body through several pathways. One pathway metabolizes ethyl alcohol to ethyl glucuronide (EtG), and another to ethyl sulfate (EtS). Normally when alcohol is metabolized in the body, most is eliminated by enzymes that are part of the glycolysis pathway that converts sugar to cellular energy. An enzyme associated with the pathway, alcohol dehydrogenase, "takes away" electrons (oxidation) from the alcohol molecules, which results in final products of water and carbon dioxide and gives no information regarding alcohol consumption. EtG and EtS, however, are formed by "nonoxidative" pathways that, instead of breaking down the alcohol molecules, make them more complex by combining them with larger molecules, which makes it nontoxic. These nonoxidative metabolites, in the case of alcohol, EtG, and EtS, are then slowly eliminated in the urine, making them excellent markers for recent alcohol consumption (1). 

The presence of EtG or EtS in urine is indicative that alcohol was consumed within the previous 3–4 days. Thus, EtG and EtS levels are a more accurate indicator of alcohol consumption over the course of several days, which makes the approach more amenable to long-term monitoring versus direct blood alcohol measurements. The detection of the combination of EtG and EtS in parallel offers greater sensitivity and accuracy for determination of recent ethanol ingestion than by detection of either biomarker alone. Finally, the identification of both EtG and EtS in a patient sample ensures that ethanol was ingested and was not produced as a result of 

Methods to detect EtG and EtS include immunoassay, gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS). Immunoassays have limited specificity and sensitivity with a limit of detection of approximately 1000 ng/mL. GC–MS offers more specificity than immunoassays with detection limits of approximately 500 ng/mL. LC–MS methods have improved specificity and detection limits to less than 

For alcohol, EtG and EtS cutoffs have become controversial because of the large number of widely used antimicrobial products (that is, gel hand cleaners) that contain alcohol. As a result, most regulatory entities have set cutoffs for EtG between 100 ng/mL and 1000 ng/mL and for EtS between 25 ng/mL and 100 ng/mL for reporting of an adverse finding. Our goal was to develop a specific and sensitive LC–MS-MS method for detecting EtG and EtS that fulfills these detection cutoffs.

All reagents and solvents were high performance liquid chromatography (HPLC) grade or analytical grade. HPLC-grade methanol and acetonitrile were purchased from Honeywell, Burdick & Jackson (Muskegon, Michigan). Purified water (Milli-Q, EMD Millipore, Billerica, Massachusetts) was used for reagent, sample, and mobile phase preparation. Ethyl  -D-glucuronide (EtG) and ethyl  -D-glucuronide-d5 (EtG-d5) were purchased from Cerilliant (Round Rock, Texas). Ethyl sulfate and ethyl sulfate-d5 (EtS-d5), were purchased from Athena Environmental Sciences, Inc. (Baltimore, Maryland). Ammonium formate (reagent grade, 97%) was purchased from Sigma-Aldrich (St. Louis, Missouri). Certified drug negative normal human urine was purchased from UTAK Laboratories, Inc. (Valencia, California). 

The individual deuterated internal standard (IS) working solutions (100 µg/mL for EtG-d5 and 50 µg/mL for EtS-d5) were prepared in methanol and stored at 4 °C until use. A 5-µL aliquot of each internal standard was added to a 0.5-mL final diluted human urine specimen, yielding final urine IS concentrations of 1000 ng/mL for EtG-d5 and 500 ng/mL for EtS-d5. For calibrators, a stock solution of 1 mg/mL of each analyte was prepared in acetonitrile and stored at 4 °C until use. A working solution of 100 µg/mL was prepared by dilution with methanol. A calibration curve (25, 50, 100, 250, 500, 1000, and 2000 ng/mL) was generated by spiking different aliquots of the working stock solution into a blank drug negative normal human urine specimen.  

Three quality control (QC) samples were prepared in triplicate with a different lot of working standard solution to yield QC concentrations of 80, 400, and 800 ng/mL in blank drug negative normal human urine specimens, respectively. The samples were prepared for analysis using "dilute and shoot" methodology.  

 Figure 1: EtG and EtS analysis (standard spiked in 1:10 diluted human urine; standard concentrations = 2000 ng/mL). Peaks: 1 = EtG, 2 = EtS. 			

LC–MS analysis was performed on an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, California) interfaced with an API 4000 MS system with electrospray ionization (ESI) (AB Sciex, Foster City, California) operated in multiple reaction monitoring (MRM) negative ionization mode (ESI–). The HPLC column used for all analyses was a 100 mm × 2.0 mm Synergi 2.5-µm Hydro-RP (Phenomenex, Torrance, California). Mobile phase A was 10 mM ammonium acetate, pH 7.6, in water and mobile phase B was a 50:50 mixture of methanol and acetonitrile. The flow rate was 0.5 mL/min. A gradient method was used from 0 to 80% B in 4 min followed by a 3-min equilibration. Urine samples were diluted 10-fold with mobile phase A before injection, and the sample injection volume was 10 µL. The HPLC column was maintained at 25 °C. For MS detection various parent–daughter transitions were evaluated (for EtG 221.2: 75.0 [EtG-1] or 221.2:85.1 [EtG-2] and for EtS 124.9:80.1 [EtS-1] or 124.9:97.0 [EtS-2]) to obtain the best sensitivity free of matrix interference. 			

The use of a small-particle polar functional HPLC phase allowed for a fast elution of EtG and EtS at less than 2 min (Figure 1) using a near-neutral mobile phase. In ESI negative mode, EtG and EtS were detected by monitoring the mass transitions and their deuterium-labeled internal standards. Quantitative and qualitative data were generated for both transitions of EtG (EtG-1 and EtG-2) and EtS (EtS-1 and EtS-2). Based on the instrumentation used and samples analyzed, one can determine the transition that works best for specific application requirements.  

Figure 2: Extracted ion chromatograms for EtG, EtS, and their internal standards (EtG-d5 and EtS-d5) at a 100-ng/mL concentration in 1:10 diluted human urine.			

The optimized HPLC method utilized a 7-min separation that included a gradient from a 100% aqueous solution to 80% organic in 4 min (Figure 2) followed by a 100% aqueous equilibration. The retention times of EtG and EtS are 1.10 and 1.30 min, respectively. Although the total run time is 7 min, the method's 2-min data window allows for multiplexing of HPLC runs to increase sample throughput and reduce MS idle time. This can be advantageous in a clinical setting where large numbers of samples need to be analyzed.  

Figure 3: Patient urine profiles: (a) Patient 2 (EtG and EtS concentrations are at relatively low levels); (b) Patient 3 (EtG and EtS concentrations are at relatively medium levels); (c) Patient 6 (EtG and EtS concentrations are at very high levels). 			

The highly polar nature of EtG and EtS made typical LC methods difficult to achieve adequate retention using traditional C18 or phenyl phase columns. In addition, these polar analytes require loading conditions utilizing close to 100% aqueous mobile phase to achieve adequate retention and separation from matrix contaminants. High aqueous mobile phase reduces MS sensitivity and raises concerns of "phase collapse" phenomena that could compromise method reproducibility. The polar endcapped C18 column used for this method provides improved polar selectivity for adequate retention of EtG and EtS and yields clear separations away from matrix contaminants. EtG is clearly separated from a potential isobaric matrix component. The method is very rugged and demonstrates adequate selectivity even for concentrated standards (Figure 1) and high-level patient samples (Figure 3c). Standard calibration curves were generated over the concentration range of 25–2000 ng/mL by plotting the relative response (peak area of EtG and EtS divided by the peak area of internal standards) versus concentration. A linear fit with weighting factor of 1/

 was used to counter the undue influence of high concentrations of EtG and EtS in the high calibrators. The standard calibration curves were linear over the calibration ranges with 

  values of 0.9989 for EtG-1 (Figure 4a), 0.9978 for EtG-2 (Figure 4b), 0.9969 for EtS-1 (Figure 4c), and 0.9959 for EtS-2 (Figure 4d).  

Figure 4: Standard curves from 25 ng/mL to 1000 ng/mL for EtG and EtS in human urine: (a) EtG-1, (b) EtG-2, (c) EtS-1, and (d) EtS-2.			

Sensitivity of the method was evaluated by determining the lowest level concentration with a signal-to-noise ratio of at least 10:1 for limit of quantification (LOD). At the lowest level standard concentration (25 ng/mL) the signal-to-noise ratios were 47.5 for EtG-1, 48.5 for EtG-2, 88.2 for EtS-1, and 205.3 for EtS-2 (Figure 5 and Table I). Thus, the LOQ values were estimated to be <25.0 ng/mL for EtG and EtS (Table I) which is well below the requirements for this assay. QC samples were prepared at levels of 80, 400, and 800 ng/mL. These concentrations were selected to represent low, medium, and high concentration across the calibration range for each analyte. The QC samples were diluted in the same way as the actual sample described above and analyzed in triplicate to assess reproducibility. The mean expected recovery of the lowest-level QC samples (80 ng/mL) were 118% for EtG-1, 103% for EtG-2, 113% for EtS-1, and 112% for EtS-2 (Table I). The percentage coefficients of variation (%CV) for the intra-assay precision ranged from 1.83% to 9.97% for levels of 80 ng/mL, 4.58 to 7.20% for levels of 400 ng/mL, and 0.98% to 2.33% for levels of 800 ng/mL (Table I). No endogenous signal was found in three normal urine specimens, which demonstrated the selectivity of the method. There was no carryover observed when blank urine samples were injected after the highest calibrator (2000 ng/mL). This optimized LC–MS procedure allows for the best recovery of the urinary EtG and EtS, and demonstrates good potential for high-throughput sample preparation and automation. 

Figure 5: Extracted ion chromatograms: (a) EtG-1, (b) EtG-2, (c) EtS-1, and (d) EtS-2.			

A rapid analysis of urinary EtG and EtS was developed that is rugged and accurate and can be easily automated and multiplexed for a high-throughput environment. The use of a polar endcapped C18 phase for this separation provided a shorter chromatographic analysis time and still separated both the EtS and EtG analytes away from matrix contaminants, which is of great importance for such "dilute-and-shoot" methods. In addition, by employing multiple parent–daughter ion transitions, the method provides additional flexibility based on the samples being analyzed and instruments being employed in the method. 

Table I: Statistical data for EtG and EtS in urine obtained by LCâMS

 are with Phenomenex Inc., Torrance, California.

(1) R.L. DuPont, G.E. Skipper, and W.L. White. 

Articles

A method is illustrated for the simultaneous analysis of ethyl glucuronide and ethyl sulfate in human urine samples.

A Sensitive, Specific, Accurate, and Fast LC–MS-MS Method for Measurement of 42 Ethyl Glucuronide and Ethyl Sulfate in Human Urine

One of the biggest debates in clinical medicine currently revolves around understanding the proper levels of vitamin D in patients' plasma. Besides well-known diseases like rickets and osteomalacia, vitamin D deficiency has been linked to cancer and heart disease and is rapidly becoming the most widely used liquid chromatography–mass spectrometry (LC–MS)–based clinical test. Increases in testing frequency have required more rapid and cost-effective solutions for determining vitamin D levels in plasma. An LC–MS method for vitamin D

 was adapted for use with core-shell columns to achieve run times of less than 4 min. This method provides rapid, sensitive, rugged, and robust LC–MS-MS analysis of vitamin D levels in patient serum (LOD of 1 and 2 ng/mL for 25-OH D

, respectively, with CV of 4–7%) and speeds the diagnosis by hospital and clinical laboratories of potential vitamin D deficiencies.

Vitamin D is recognized as an essential nutrient with its primary physiological function being to increase intestinal absorption of calcium and phosphate and to promote deposition of these minerals in newly formed bones. Deficiency and abnormal vitamin D levels result in impaired bone mineralization and lead to bone softening diseases — rickets in children and osteomalacia in adults. In addition, a large number of bone disorders and mineral metabolism defects have been associated with abnormal vitamin D levels, including nephritic syndrome, granulomatous diseases, and hypocalcemia, as well as secondary hyperparathyroidism, which frequently complicates renal failure. Additional well-known maladies, including cardiovascular disease, cancer, and autoimmune disorders, recently have been found to be influenced by vitamin D deficiencies; as a result, vitamin D testing has increased tremendously across a variety of patient groups (1,2). 

Vitamin D is metabolized to 25-OH D in the liver. Total vitamin D is best determined by measuring total 25-hydroxyvitamin D (D

) in serum because the half-life of 25-OH D is about three weeks with serum concentrations of 10–50 ng/mL. Vitamin D supplementation in both food and tablets comes in both the D

 forms, making it imperative to measure 25-OH D

. Although optimal serum concentrations of total 25-OH D are generally agreed to be ≥30 ng/mL, there is considerable discussion on the serum concentration of 25-OH D considered to be inadequate for bone and overall health, but <20 ng/mL is generally regarded as deficient. Serum concentrations >100 ng/mL are generally regarded as potentially toxic (3). 

Vitamin D exhibits a high propensity for inherent endogenous serum protein binding and association. Vitamin D is typically not found free in serum samples, thereby posing a challenge for sensitive and reproducible high performance liquid chromatography (HPLC) analysis without appropriate sample preparation. Additionally, various serum sample matrix constituents are found to cause ion suppression that reduces accuracy and reproducibility during patient sample analysis. This ion suppression can be especially troublesome with rapid LC–mass spectrometry (MS) methods where insufficient resolution can be observed between matrix contaminants and analyte peaks. 

A recent innovation in HPLC column technology has been the introduction of core-shell silica particles. Unlike fully porous silica media, core-shell media employ structured particle geometry where a thin porous shell is grafted on a nonporous core particle. This particle geometry has a reduced analyte diffusion distance along with a tight particle size distribution that results in column efficiency on par with or better than sub-2-µm media while maintaining column back pressures more in line with traditional HPLC columns. Higher performance at lower back pressure using core-shell HPLC columns offers the promise of improving existing HPLC methods without having to replace existing HPLC instrumentation with ultrahigh-pressure-compatible LC systems. This article discusses an example of using core-shell columns to improve the resolution of a separation while reducing analysis run time. 

 were obtained from Medical Isotopes (Pelham, New Hampshire) and 25-hydroxyvitamin D

 was obtained from Sigma-Aldrich (St. Louis, Missouri). HPLC-grade water (Milli-Q, Millipore, Billerica, Massachusetts) was used to prepare HPLC mobile phase and for sample preparation. Methanol and acetonitrile were obtained from Honeywell, Burdick & Jackson (Muskegon, Michigan). Stock internal standard solution was prepared by dissolving the contents of a 5-mg vial of 25-hydroxyvitamin D

 in 5.0 mL of ethanol. The precipitation reagent was prepared by adding 10 µL of internal standard stock solution to 60 mL of 95:5 (v/v) acetonitrile–methanol in a 100-mL volumetric flask and diluting to volume with 95:5 acetonitrile–methanol. To analyze patient samples, 350 µL of precipitating reagent containing internal standard was combined with 100 µL of serum and vortexed for 30 s. After inspecting the tube to ensure that the sample was fully mixed, the sample was centrifuged at 13,000 rpm for 15 min. Supernatant was transferred to an HPLC vial and 40 µL was injected onto an LC–MS-MS system. 

LC–MS-MS was performed using an HPLC system equipped with binary pump, autosampler, and column oven interfaced with an Applied Biosystems 4000 Qtrap mass spectrometer (AB Sciex, Foster City, California) with a Turbo V ion source or with an Applied Biosystems API 4000 triple-quadrupole mass spectrometer. An atmospheric pressure chemical ionization (APCI) source was used and was run in positive ion mode. The MS-MS operating conditions are listed in Table I. The HPLC column used for all analyses was a 50 mm × 4.6 mm, 2.6-µm core-shell Kinetex C18 column (Phenomenex, Torrance, California) operating at a flow rate of 1 mL/min. This method was compared against a methodology using a fully porous 5-µm C18 column. A gradient method was employed using an aqueous mobile phase A of 0.05% formic acid in water and an organic mobile phase B of 0.1% formic acid in methanol with 5 mM ammonium acetate. The gradient was as follows: hold at 8% B for 5 s, then 8–100% B in 200 s (3.33 min). The column was washed and re-equilibrated between injections and maintained at 35 °C. 

 from serum necessitates the use of sample preparation procedures to remove potential matrix constituents, which will interfere with accurate and precise determination of 25-OH D in serum and reduce HPLC column life. Vitamin D and the hydroxy D metabolites are relatively more hydrophobic than virtually all endogenous and exogenous compounds that are typically quantified in biological matrices. The propensity for vitamin D to inherently associate with serum proteins reduces the bioavailability of free vitamin D in serum; therefore, it is necessary to disrupt this association for improved assay accuracy and precision. Protein precipitation is the easiest means of disrupting the serum protein association with hydrophobic analytes. In this method, protein precipitation is performed in 1.5-mL centrifuge tubes with the addition of 100 µL of serum sample to 350 µL of 95:5 acetonitrile–methanol containing the 25-OH D

 internal standard. The low solubility of the endogenous proteins in acetonitrile results in their precipitation from the sample; mixing and centrifugation cause the precipitated protein to form a pellet at the bottom of the centrifuge tube, and the supernatant is then analyzed. An alternative to the traditional protein precipitation approach involves the use of a protein precipitation plate, such as Strata Impact (Phenomenex), which contains an oleophobic membrane filter. Following addition of acetonitrile to the serum sample to facilitate protein precipitation, the sample passes through a 0.2-µm cut-off filter of the precipitation plate to eliminate the precipitated protein from the sample. 

Table II: API 4000 triple-quadrupole MS system 			

LC–MS was performed in multiple reaction monitoring (MRM) mode (see Tables II and III for the MRM transitions monitored using the API 4000 and QTRAP MS systems, respectively). The use of MRM is important because 25-OH D

 are not separated chromatographically; however, the unique parent–daughter ion combination for each analyte allows for specificity and accurate determination of the concentration for each analyte in the sample. 25-OH D

 was used as an internal standard and signal intensity of each 25-OH D analyte relative to the internal standard was used for determining the concentration of 25-OH D

 in the sample. The calibration curves were linear over the range from 0 to >500 ng/mL, with observed limits of detection (LOD) of 1 and 2 ng/mL for 25-OH D

, respectively. The reproducibility of this assay was very good with CV of 4–7%. 

Both the HPLC and UHPLC conditions using a 50 mm × 2.0 mm, 5-µm fully porous C18 column and the 2.6-µm core-shell column, respectively, allow for sufficient retention of 25-OH D

, further minimizing the potential for interference and ion-suppression from any weakly retained impurities (Figures 1 and 2). Using the 5-µm C18 column, elution of 25-OH D

 occurred in just under 5 min, with an overall chromatographic run time of 8 min, including column re-equilibration. The separation using the 2.6-µm core-shell C18 column is similar; however, 25-OH D

 are eluted in less than 4 min. This allows the overall chromatographic run time, including column re-equilibration, to be reduced to 6 min. The shorter analysis time using the core-shell column is a significant benefit for laboratories analyzing a large number of samples in a high-throughput sample environment — the reduction in overall chromatographic run time translates into a 25% increase in sample throughput and corresponding reduction in solvent usage. In addition to the faster chromatographic separation, the peak intensities are significantly larger on the core-shell column resulting in improved sensitivity and lower quantitation limits. 

Figure 1: LCâMS-MS analysis of 25-OH D2 and D3 standards run on a fully porous 5-Âµm C18 column. Note the similar elution time for 25-OH D2, 25-OH D3-d6 internal standard, and the 25-OH D3 peaks. 				

Figure 2: UHPLCâMS-MS analysis of 25-OH D2 and D3 standards run on a Kinetex 2.6-Âµm C18 column. Note the reduced retention time, improved peak shape, and increased peak height for the standards run on the Kinetex core-shell column. 			

The most important benefit is the improved efficiency and resolution provided by the core-shell column. With the fully porous 5 µm C18 column, an endogenous compound (D

 cholesterol) present in patient samples was found to be coeluted with the 25-OH D

 peak, impacting accurate quantitation and increasing the need to reanalyze a large percentage of patient samples (Figure 3). The presence of the endogenous compound in patient samples was resolved from 25-OH D

 and was eluted outside the quantitation window on the core-shell column (Figure 4), resulting in increased analytical accuracy. This improvement in throughput and resolution was achieved at system back pressures compatible with standard HPLC systems (400-bar back-pressure limit). 

Figure 3: LCâMS-MS analysis of 25-OH D2 and D3 standards spiked into plasma run on the fully porous 5-Âµm C18 column after protein precipitation. Note the interfering D3 cholesterol peak that is eluted just after the 25-OH D3 peak. This interference can greatly perturb the quantitation of the D3 peak. 			

The analytical method presented above allows for the rapid and accurate determination of vitamin D levels in patient serum samples and speeds the diagnosis of potential vitamin D deficiencies indicative of specific disease states quickly and with a high degree of precision. Protein precipitation is used for sample preparation of patient serum samples, effectively disrupting the serum protein association with 25-OH D

 and providing sufficient sample cleanup before LC–MS-MS analysis. Protein precipitation plates would be an effective alternative for sample preparation in a high-throughput clinical laboratory environment. 

Figure 4: LCâMS-MS analysis of 25-OH D2 and D3 standards spiked into plasma run on the core-shell Kinetex 2.6-Âµm C18 column after protein precipitation. Note that the interfering peak in Figure 3 with the fully porous column is not eluted near the 25-OH D3 peak, thus peak quantitation is not affected when the core-shell column is used for this application.  			

The core-shell column allows for faster chromatographic analysis of patient samples and increased signal intensity for improved sensitivity. The increase in chromatographic resolution from an endogenous compound present in patient serum samples provides improved accuracy and reproducibility and a decrease in sample reanalysis.  This clinical example shows the advantages of using core-shell columns with existing HPLC instrumentation to realize an improvement in throughput and performance. Other UHPLC columns require ultrahigh-pressure instrumentation, which can be a difficult (and unnecessary) choice in the current economic climate. 

 are with Phenomenex, Torrance, California.  

(1)  Dietary Supplement Fact Sheet: Vitamin D; Office of Dietary Supplements, National Institutes of Health (

http://ods.od.nih.gov/factsheets/vitamind.asp

(3) Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes: Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press, 1997. 

Increases in vitamin D testing frequency have required more rapid and cost-effective solutions for determining vitamin D levels in plasma. This LC–MS method was adapted for use with core-shell columns to achieve run times of less than 4 min.

25-Hydroxyvitamin D2/D3 Analysis in Human Plasma Using LC–MS

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.

Michael McGinley

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