Liming Peng

Automated solid-phase extraction (SPE) has been used extensively with liquid chromatography–tandem mass spectrometry (LC–MS-MS) to facilitate high-throughput analysis in the pharmaceutical, diagnostic, and forensic toxicology areas. In this work, we demonstrate the use of a systemized approach to SPE method development and LC–MS-MS analysis. This approach provides dramatic savings in analysis time and takes advantage of new innovations in high performance liquid chromatography (HPLC) columns to provide the cleanest extracts for LC–MS injection.

The use of automated solid-phase extraction (SPE) has been shown to reduce sample preparation time by at least 50% in comparison with manual SPE (1). A further 50% improvement in throughput can be achieved by avoiding evaporation and reconstitution steps and direct injecting the SPE extracts into the liquid chromatography–mass spectrometry (LC–MS) system (1). An additional advantage with direct injection of SPE extracts is elimination of analyte loss during evaporation, as observed for compounds such as isoniazid (2) and indomethacin (3). 

For the cleanup of basic drugs, associated SPE methods commonly utilize strong cation exchange sorbents. However, even acidic and neutral compounds can be cleaned up effectively with these strong cation exchange sorbents, including hydrophobic compounds such as THC-carboxylic acid or very polar molecules like acrylamide, γ-amino butyric acid analogs, and antivirals like penciclovir (4–7). 

The preference for employing strong cation exchange sorbents also stems from the fact that proteinaceous and endogenous components of biological matrices can be washed out very efficiently with a strong organic solvent before elution of basic analytes. Strong organic washes furnish very clean elution extracts, displaying insignificant ion suppression and enhancing lower detection and quantitation limits in analyses (8). A vast majority of drugs are basic and fit this scenario perfectly.  

The typical elution conditions used during SPE of biological samples with strong cation exchange sorbents consist of an organic solvent containing 2–5% ammonium hydroxide (9,10). 

The nature of the strong organic elution solvent and the high pH of these extracts have made such elutions incompatible with traditional column chemistries and reversed-phase solvent conditions. As such, the common practice is to evaporate off the ammonia along with the solvent and reconstitute the analyte or analytes in an aqueous organic solvent amenable to reversed-phase LC–MS analysis. 

The recent development of LC columns that have increased pH stability gives method developers the option to inject these high-pH extracts directly into the LC–MS-MS system. Studies during the last decade clearly show that LC–MS employing high-pH mobile phases on the novel stationary phases, resistant to degradation/dissolution under such conditions, furnishes several advantages (11,12). Most attractive are the enhancement in sensitivity and resolution, along with improved retention for polar basic analytes. These investigations also show that mixtures of neutral, acidic, and basic analytes can be analyzed efficiently under these high-pH mobile phase LC–MS conditions, which when extrapolated to drug–metabolite combinations are highly desirable for obtaining excellent resolution and sensitivity for very low abundance metabolites.  

A multisorbent strata-X method development plate and single-sorbent strata-X-C 96-well plates (both with 10-mg bed masses) were obtained from Phenomenex (Torrance, California). All the analytes studied were obtained from Sigma (St. Louis, Missouri) and used as such. Human plasma was obtained from Valley Biomedical (Winchester, Virginia).  

A pH-stable 50 mm × 2.0 mm Gemini NX C18 column was obtained from Phenomenex. MS detection was done using an ABI 3000 mass spectrometer with electrospray ionization in the positive ion mode at a temperature of 425 °C (Applied Biosystems, Foster City, California). 

All reagents and solvents used were also from Sigma and were used without further purification. 

Stock solutions of atenolol, toliprolol, bunitrolol, and bupranolol were made by dissolving each drug in methanol at a concentration of 1 mg/mL. The plasma samples were spiked with appropriate volumes of these solutions corresponding to concentrations of 0.5–500 ng/mL of plasma. The concentration of nadolol (internal standard) was 40 ng/mL. 

Automated SPE Method Development Protocol

Step 1: Method development with the strata multisorbent well plate: 

The strata-X method development plate was conditioned with 400 μL of methanol followed by 400 μL of water. The plasma solutions were loaded under the three sets of conditions specified below:   

NN: neutral load and wash-1 in water and elute with methanol 

AB: Load and wash-1 in 25 mM ammonium formate buffer (pH 2.5) and elute with 5% ammonium hydroxide in methanol 

BA: Load in 25 mM ammonium acetate (pH 5.5) and elute with 2% formic acid in methanol)

After the initial wash (wash-1) at one specific pH, the second wash (wash-2) was done with 400 μL of 70% methanol before elution. The plate was dried for 1 min at 10 in. of Hg before elution. 

Step 2: Method validation with the protocol developed in Step 1:  

The single-sorbent well plate was conditioned with 400 μL methanol followed by 400 mL of 0.1 M acetic acid. Human plasma with disodium EDTA as anticoagulant was spiked with analytes (studied for a wide calibration range for precision and accuracy) and internal standard (nadolol). Samples were diluted (1:2) with 0.1 M acetic acid before loading on the plate. Washing was done in two steps, first with 400 μL of 0.1 M acetic acid, followed by 400 μL of methanol. Before eluting with 300 μL of 5% ammonium hydroxide in methanol, in two increments, the sorbent was dried for 60 s under high vacuum of 10 in. of Hg. The elution was injected directly into the LC–MS-MS system.  

 Gemini NX column was used under gradient conditions, using a mobile phase of 5 mM ammonium bicarbonate (pH 10.0)–acetonitrile from 90:10 to 25:75 in 2.5 min followed by equilibration for 1.5 min with a flow rate of 0.5 mL/min. The injection volume was 5 μL. 

The acidic mobile phase runs utilized the standard mobile phase conditions consisting of aqueous 0.1% formic acid and acetonitrile, with the same gradient conditions as for the basic analysis.  

Figure 1 gives the structures of the analytes and internal standard used in this study, along with their p

 and log P values. Of all the analytes, atenolol is the most polar, though all of them display about the same basicity. All analytes are secondary amines. 

Figure 1: Structures and physicochemical parameters of the probes studied. 

The first step in an overall analytical method development process for an analyte from any matrix, and a biological matrix in particular, is to develop an appropriate sample preparation protocol. The purpose of sample preparation is to concentrate the analyte and to clean it up from matrix constituents that can interfere with its analysis. Sometimes, a selective separation–enrichment is a bonus, as with low-abundance metabolites in large quantities of drugs.  

To simplify the method development process, we designed an automated SPE method development protocol using a multisorbent 96-well plate in tandem with an automatic liquid handler (13). This well plate consists of four different functionalized polymeric sorbents, a neutral sorbent (strata-X), a strong cation exchanger (strata-X-C), a weak cation exchanger (strata-X-CW), and a weak anion exchanger (strata-X-AW). These four collectively cover a wide range of polarities and cater to all possible modes of hydrophobic and polar interactions (including ion exchange interactions) that can be displayed by any analyte.  

The Step-1 generic protocol was designed for screening all four sorbents simultaneously using three sets of conditions, as outlined in the Experimental section. After this Step-1 protocol is completed, a method developer is able to identify the most appropriate sorbent, as well as the load, wash, and elution conditions (pH and solvent) for the subsequent validation step (Step 2).  

In Step 1, a 70% methanol wash step was utilized after an initial water or buffer wash. From the work of Chambers and colleagues (8), it is evident that methanol is an excellent solvent for the removal of phospholipids and other endogenous materials from plasma, even at concentrations of 60–80%.  

Aimin Tan and coworkers (14) report a protocol that takes advantage of the fact that basic analytes undergo a dramatic reduction in the degree of ionization at or near their p

 values, which enables the hydrophobic mechanism to operate more effectively with neutral sorbents that permit a stronger organic wash. The results from Step-1 protocol (Figure 2) demonstrate that even without employing any such basic aqueous–organic wash conditions, the neutral sorbent provides recoveries of around 60% for the strongly polar basic analyte atenolol when a wash-2 with 70% methanol is used. No loss of atenolol was observed when 30% methanol was used for wash-2 with the neutral sorbent. 

Figure 2: Recovery of atenolol from a strata multisorbent 10-mg 96-well plate. 

Even though decent recoveries were obtained with the neutral sorbent under the NN conditions, the results of the Step-1 program indicated that the strong cation exchange sorbent (strata-X-C) yielded the best results of any of the sorbents using the AB conditions. Recoveries of 98% were achieved when samples were subjected to a 70% methanol wash (Figure 2). The same near-quantitative recoveries were obtained with 100% methanol wash, allowing for efficient removal of phospholipids and other endogenous materials, which are responsible for ion suppression during LC–MS-MS. However, the matrix effects were insignificant with the 100% methanol wash.  

Step 2 incorporates the optimized conditions developed in Step 1, except that 100% methanol wash was used and a 96-well strong cation exchange plate is used for validation of the method. Of particular interest in this analysis was the use of the AB conditions. Under these conditions, the eluates will be in 5% ammonium hydroxide in methanol. It is common practice to evaporate off the ammonia along with the solvent and reconstitute the residue in a formic acid–acetonitrile mobile phase for LC–MS-MS analysis, as reported in all documented literature using strong cation exchange sorbents for sample preparation.  

This evaporation–reconstitution for 96 samples involves an additional 2.5–3.0 h that laboratories must spend to prepare their samples. However, if it is possible to avoid these steps and inject the ammonia–organic extract directly into the LC–MS-MS system, this time could be saved. Due to 100% methanol wash, the eluate from the strong cation exchange material is extremely clean, so there is less potential for contamination. An additional advantage of the direct injection option is that basic drugs will be in either neutral or near-neutral form (depending upon their p

 values), and this would enhance hydrophobic interactions and hence, retention under reversed-phase high performance liquid chromatography (HPLC) conditions.  

However, such direct injection onto traditional silica-based reversed-phase columns is not feasible because of stability problems. The recently introduced Gemini and Gemini-NX columns allow method developers to use pH conditions from 1–12. Therefore, handling the high-pH mobile phases in the present work with Gemini-NX was not problematic.  

Previous work from our laboratory has demonstrated that when performing LC–MS-MS analysis using these new materials in high-pH mobile phases, analyte sensitivity can be increased (11). The increase in sensitivity achieved by performing chromatographic separations at high pH is due to multiple factors, including extended retention, excellent peak shapes, and good efficiency. However, this increase in sensitivity is observed not only for the basic analytes, but for some acidic and neutral analytes as well.  

Figure 3: Linearity of toliprolol and atenolol over a 1000-fold concentration range. 

The data collected from Step 2 through LC–MS-MS analysis under mobile phase conditions outlined in the experimental section is summarized in Figures 3–5. Figure 5 shows a representative chromatogram for these drugs in both acidic and basic mobile phase conditions. In comparison with data from the same extracts run under acidic conditions, employment of the high-pH mobile phase showed about 1.5–4.0 times higher sensitivity for the four analytes studied. This increase in sensitivity helped compensate for the fact that we did not concentrate extracts through evaporation and reconstitution steps. Also interesting was the fact that the peak shape of all compounds, including atenolol, was not affected adversely due to the high organic concentration of the injection solvent, under either basic or acidic conditions. More will be published on these results at a later date. 

Figure 4: Linearity of bunitrolol and bupropranolol over a 1000-fold concentration range. 

Figures 3 (atenolol and toliprolol) and 4 (bunitrolol and bupranolol) show the linearity of the method over the concentration range studied, 0.5–500 ng/mL for these analytes from the Step 2 protocol. The low limit of quantitation, broad dynamic range, and precision obtained suggest that this method would be suitable for most pharmaceutical and other analytical needs. 

Figure 5: Comparison of sensitivity between high pH (a) and low pH (b) mobile phases for atenolol, nadolol, and bunitrolol.  

The use of a structured SPE method development protocol quickly identified the best sorbent for the extraction procedure. The use of the strong cation exchange material allowed for 100% methanol wash conditions, resulting in extremely clean extracts. Using the Gemini-NX high pH stable HPLC column allowed the basic eluate to be injected directly onto the LC–MS-MS system. The high-pH chromatographic conditions resulted in higher MS response. By eliminating time-consuming steps throughout the process, the resulting method is perfectly suited to the high-throughput environment. 

Although this work demonstrated the advantages of combining automated sample preparation with direct injection and high-pH LC–MS-MS analysis for a small set of basic probes, this universal protocol is applicable to any analyte, neutral, acidic, or basic, from any kind of matrix. 

Liming Peng, Shahana Wahab Huq, Krishna M. Kallury

 are with Phenomenex, Torrance, California.  

Articles

Here, the authors demonstrate the use of a systematized approach to SPE method development and LC–MS-MS analysis.

A Simplified Approach to Optimize SPE Method Development with Downstream LC–MS Analysis Allowing 100% Organic, Basified Injection Solvents

Beta-blockers are basic compounds that contain a secondary amino group in their structure. The amino substituents are typically an isopropyl group and a larger chain with a hydroxyl group in the beta position from the nitrogen atom (Table I). The simultaneous analysis of β-blockers in biological samples is meaningful, and is made possible by the similarities in their structure. Gas chromatography (GC)–mass spectrometry (MS) has been the most used technique for their identification and quantification (4–6). However, most β-blockers are nonvolatile and thus require derivatization via a cumbersome and time-consuming process before GC–MS analysis. In recent years, liquid chromatography (LC) coupled with mass spectrometric detection has evolved as the method of choice for drug analysis in the pharmaceutical, clinical, and forensic toxicology areas (4–8). In contrast to GC–MS, LC–MS-MS generally does not require derivatization and offers superior sensitivity. Moreover, due to the high specificity offered by LC–MS-MS, baseline chromatographic resolution often is not required, allowing for fast analysis in high-throughput environments. 

In this work, a high-speed reversed-phase LC positive ion mode electrospray tandem mass-spectrometric (LC–ESI-MS-MS) method for the quantitative determination of nine β-blockers — acebutolol, alprenolol, atenolol, labetalol, metoprolol, pindolol, propranolol, sotalol, and timolol — in human plasma is presented. Solid-phase extraction (SPE) cartridges are used for sample cleanup and a C18 high performance liquid chromatography (HPLC) column for separation in high-pH mobile phase. The novelty of our approach resides in the choice of mobile phase. Most often, basic compounds are analyzed by LC–MS-MS in acidic mobile phase in which the analytes are positively charged. Unfortunately, such conditions are not favorable for reversed-phase chromatographic separations because charged basic species are poorly retained on hydrophobic sorbents. Polar bases are the biggest challenge for several reasons: they are eluted in the early region of the chromatogram, where ion suppression tends to be most problematic, peak shapes are poor, and peak capacity is typically low. In high-pH mobile phases, basic compounds are ionized only partially (if at all), behaving more like neutral species. They are retained very well due to more favorable hydrophobic interaction, and are eluted with narrow and symmetrical peaks. A further benefit to our choice of mobile phase is the positive effect on the MS detection of β-blockers. 

An Agilent 1100 series (Wilmington, Delaware) HPLC system comprised of a G1312A binary pump and a G1329A autosampler module was coupled to an Applied Biosystems (Foster City, California) API 3000 triple-quadrupole mass spectrometer provided with TurboIonSpray ion source operated in positive ion mode (PIM) with multiple reaction monitoring (MRM). The source heater temperature was set to 425 °C and the heater gas flow to 7000 cc/min. 

Chromatographic separations were performed on the extended pH-range HPLC column Gemini (Phenomenex) 3 μm C18 with the dimensions 50 mm × 2.0 mm. The column was operated at a flow rate of 0.5 mL/min in gradient mode. A quantity of 5 mM ammonium bicarbonate (NH

) in water (pH 10.0) was used as mobile phase A, with acetonitrile as mobile phase B for high-pH experiments. Mobile phase A was 0.1% formic acid in water (pH 2.7), and 0.1% formic acid in acetonitrile was used as mobile phase B for low-pH experiments. Gradient elution was started at 10% B and the mobile phase composition increased to 75% B in 2.5 min. The column was reequilibrated for 1.5 min. This gradient profile was used at both low and high pH. The injection volume was 5 μL. 

A 200 μL aliquot of human plasma was diluted with 500 μL of water and 100 μL of 1 M acetic acid. A 100 μL volume of 20 ng/mL oxprenolol was added as internal standard, along with 200 μL of a standard solution of β-blockers (in water) at the following concentration levels: 0.1, 0.5, 1.0, 10, 100, 500, or 1000 ng/mL, respectively. The concentration of the internal standard in the sample was 10 ng/mL. 

Sample cleanup before chromatographic analysis was performed using SPE with the following procedure: strata-X-C 30 mg/1 mL cartridges (Phenomenex) were conditioned with 1 mL of methanol, followed by 1 mL of water. The sample was loaded and first washed with 1 mL of 0.1 M acetic acid solution followed by 1 mL of methanol. Analytes were eluted using 1 mL of 5% ammonium hydroxide (prepared with 28.5% concentrated ammonium hydroxide) in methanol. The extract was evaporated to dryness under gentle nitrogen gas flow and then reconstituted in 200 μL of 10% acetonitrile in water. 

The Gemini 3 μm C18 110 Å high-pH stable HPLC column used in this study is well suited for the chromatographic separation of acidic, neutral, and basic compounds over an extended mobile phase pH range (1.5–12). This sorbent is made by grafting an organic protective layer onto the silica surface (9).  

Previously, we reported on the successful MS detection of basic compounds in high-pH mobile phases (10). Despite the high pH reducing analyte ionization in solution (or in some cases minimizing it, such as in mobile phases of pH ≥ p

 +2), it was shown that several of the β-blockers included in the present work provided similar signal intensities in high- and low-pH mobile phases. We reconfirm these findings for all six of the β-blocker compounds examined in the previous work and report similar results for four more, at the low concentration level of 0.5 ng/mL (Figure 1). Contrary to expectation, in this and the previous work more intense signals were observed for all 10 β-blockers (including oxprenolol, the internal standard) in high-pH mobile phase, with the most significant increase in MS response observed for sotalol and atenolol (80% and 30%, respectively) at the concentration level of 0.5 ng/mL. 

Figures 2 and 3 show a direct comparison of MS-MS responses at low pH (pH 2.7 in mobile phase containing 0.1% formic acid), and high pH (pH 10.0 in mobile phase containing 5 mM ammonium bicarbonate). At high pH, β-blockers are uncharged (free-base) in solution and therefore show better retention on reversed-phase sorbents. Suppressing analyte ionization effectively increases retention of the most polar β-blockers (such as sotalol and atenolol) and thus allows high-speed chromatographic separations.  

Biological samples should be cleaned up before LC–MS-MS analysis for two reasons: first, for preventing the buildup of matrix components on the chromatographic column (which drastically limits its life), and second, for preventing the coelution of matrix components with analytes of interest. Such coelution often causes analyte ion suppression, which can compromise the results of the analysis and lead to incorrect quantification. The polymer-based strata-X-C (Phenomenex), an SPE sorbent possessing both hydrophobic and hydrophilic properties as well as cation-exchange capability, is effective for the selective extraction of basic compounds from complex matrices. The efficacy of the SPE procedure was evaluated at two concentration levels (0.5 and 100 ng/mL), with β-blockers spiked into human plasma. Absolute recoveries were determined by comparing MRM peak areas for standard solutions and for spiked plasma extracts at the same concentration level. Analyte absolute recoveries (for the combined SPE–LC–MS-MS assay) were >59%, while precision (RSD) was <15% (interassay; 

 Ion-suppression experiments revealed suppression areas away from the elution region of all analytes, including early eluted compounds typically problematic in separations performed in low-pH mobile phase (Figure 4). This indicates that matrix effects can be minimized by carefully choosing the pH of the mobile phase and by extending the retention of polar compounds, which can be eluted away from the early region of the chromatogram when analyzed in uncharged form (in high-pH mobile phases). 

The method employed here was linear in the concentration range 0.1–500 ng/mL. Calibration samples were prepared at the levels 0.1, 0.5, 1.0, 10, 50, 100, and 500 μg/mL in human plasma with the internal standard at 10 ng/mL. Table II shows linear regression data (

 ) for nine β-blockers (for SPE combined with LC–MS-MS analysis). These results demonstrate good linearity (

 ≥ 0.996) over a wide dynamic range (≤10

The limit of quantitation (LOQ) determined as the minimum analyte amount on-column giving a signal-to-noise ratio (S/N) of 10 was 5 pg on-column or better for all β-blockers included in this study (Table III). 

Method accuracy was evaluated by comparing the mean value of six replicate experimental results with the expected value at low and high concentration levels (0.5 and 100 ng/mL). Results are shown in Table III. Intra-assay accuracy was 85–120% at low concentration levels and 90–110% at high concentration levels.  

A high-speed reversed-phase LC–ESI-MS-MS method was developed for the analysis of nine β-blockers using SPE cartridges for sample cleanup, and a C18 HPLC column for chromatographic separation within 4 min (total cycle time). 

The β-blockers included in this study were detected with good sensitivity in high-pH mobile phase (pH 10.0) containing ammonium bicarbonate buffer and acetonitrile. 

Significant matrix effects for polar β-blockers can be minimized by choosing the mobile phase pH judiciously; increased retention of polar compounds (such as polar bases in high-pH mobile phase) will move their retention time away from the early region of intense ion suppression. 

The simultaneous quantitation of β-blockers by LC–ESI-MS-MS in a mobile phase containing a high-pH buffer provides an attractive alternative for analyzing and monitoring β-blockers in biological samples in diagnostics, therapeutic monitoring, doping screening, and forensic analysis.

(1)  J.M. Cruickshank and B.N.C. Prichard, 

(Churchill Livingstone Inc., New York, 1988). 

(3)  WADA. The World Anti-Doping Code. The 2004 Prohibited List, International Standard, 2004. 

(4)  H. Ktaoka, S. Narimatsu, H.L. Lord, and J. Pawliszyn, 

(6)  K. Deventer, P. Van Eenoo, and F.T. Delbeke, 

 (9)  US Patent 2,005,035,217 (September 30, 2005). 

 are with Phenomenex Inc., Torrance, California. 

Beta-blockers are basic compounds that contain a secondary amino group in their structure. The amino substituents are typically an isopropyl group and a larger chain with a hydroxyl group in the beta position from the nitrogen atom (Table I). The simultaneous analysis of ?-blockers in biological samples is meaningful, and is made possible by the similarities in their structure. Gas chromatography (GC)–mass spectrometry (MS) has been the most used technique for their identification and quantification (4–6). However, most ?-blockers are nonvolatile and thus require derivatization via a cumbersome and time-consuming process before GC–MS analysis. In recent years, liquid chromatography (LC) coupled with mass spectrometric detection has evolved as the method of choice for drug analysis in the pharmaceutical, clinical, and forensic toxicology areas (4–8). In contrast to GC–MS, LC–MS-MS generally does not require derivatization and offers superior sensitivity. Moreover, due to the high specificity offered by LC–MS-MS, baseline chromatographic resolution often is not required, allowing for fast analysis in high-throughput environments.

Liming Peng

Articles

A Simplified Approach to Optimize SPE Method Development with Downstream LC–MS Analysis Allowing 100% Organic, Basified Injection Solvents

High-Speed Analysis of b-Blockers and Metabolites in Human Plasma by LC–ESI-MS-MS with High-pH Mobile Phase