Use of An Improved Version of C8+SCX Mixed-Mode Solid Phase Extraction Material for Clean Extraction and Recovery of Basic, Zwitterionic, Neutral and Acidic Compounds from Biological Fluids Yuhui Yang, An Trinh, Michael Ye, and Tom Henderson 595 North Harrison Road, Bellefonte, PA 16823 T404109 HKB
Abstract C8+SCX mixed-mode bonded silica is the most commonly used sample prep tool for systematic toxicological analysis. However, both zwitterionic compounds and polar basic compounds cannot be effectively extracted and recovered from most commercially available mixed-mode phases. This is primarily due to the low ion-exchange capacity inherent with phases commercially available. In this presentation, we describe the use of a mixed-mode phase in which ion-exchange capacity has been optimized for extraction of polar basic and zwitterionic compounds. Three generic extraction protocols have been determined. The first protocol offers superior selectivity when recovering non-polar to moderately polar basic and zwitterionic compounds. The second protocol is used to recover very polar basic and zwitterionic compounds. The third protocol is aimed to systematically fractionate basic and zwitterionic compounds from neutral and acidic compounds for further analysis. The novel mixedmode SPE phase offers improved performance over other commercially available silica- and polymer- based mixed-mode products.
Introduction 1. Reliable and reproducible sample preparation methodology is critical for analyzing pharmaceutical drug candidates, drugs of abuse, and their metabolites from biological matrices. 2. In systematic toxicological analysis (STA), the undirected drug screening must be a generic procedure where the substances of interest are isolated at a yield as high as possible and the biological matrixes should be removed. Only one SPE tube should be used. 3. However, most commercially available mixed-mode phases have inadequate ion-exchange capacity to retain more polar basic and zwitterionic compounds. As a result, the compounds are lost during load and wash steps during the extraction procedure. 4. In contrast, polymer-based mixed-cation phases have inadequate hydrophobic capacity and thus many substances of interest are frequently lost in the methanol wash step.
Experimentation/Results General Mixed-Mode SPE Methodology Condition with methanol & equilibrate with ph 3-6 buffer Load Sample @ low ph 3-6 Wash 1: low ph 3-6 buffer Wash 2: 100% methanol Elute with basified (high ph)methanol Sample must be aqueous to support reversed-phase component of interaction. Low ph environment ionizes basic compounds for ionexchange & neutralizes acidic compounds to promote reversed-phase interactions Wash 1 further reinforces Ion-Exchange interaction with basic compounds, neutralizes acidic compounds for Reversed-Phase retention, and removes all non-basic hydrophilic interferences Wash 2 is a powerful wash step used to remove all hydrophobic interferences. If fractionation of acidic and neutral components are necessary, both compound groups will be eluted in this step Methanol alone will not elute basic compounds due to the Ion-Exchange component of interaction. By basifying methanol, basic compounds are neutralized disrupting both electrostatic and hydrophobic interaction
Generic Extraction Protocol 1 For Non-Polar Basic and Zwitterionic Compounds (100mg/1mL or 100mg/3mL SPE tube) 1. Sample Pre-Treatment: Dilute urine (plasma/serum) sample (1.0mL) with 50mM ammonium acetate buffer or 10mM potassium phosphate buffer (ph6.0) (1.0mL). 2. SPE Tube Conditioning: Solvate tube with 1mL methanol 3. SPE Tube Equilibrium: Equilibrate tube with 50mM ammonium acetate buffer or 10mM potassium phosphate buffer (1mL) 4. Sample Loading: Load pre-treated urine or plasma (1mL) at a flow rate of 1mL/min 5. Interference Elution: Elute interference with 50mM ammonium acetate buffer (or 10mM phosphate buffer) (1mL), 1M acetic acid (1mL) and methanol (1mL). 6. Analyte Elution: Elute analytes with 5% ammonium hydroxide in methanol The generic protocol is only suitable for compounds that are ionized at ph 6 load conditions to facilitate ion-exchange interaction, and/or of sufficient non-polar character to interact with bonded phase C8 functional groups.
Case 1: Extraction of Tricyclic Antidepressants Using DSC-MCAX SPE mau Spiked Serum Sample Prior to SPE cleanup Compound (10ng/mL) % Abs Recovery ± RSD (n=3) mau -0.2 0.0 0.2 0.4 0.6 0.8 6 Spiked Serum 1 Sample After DSC-MCAX 2 cleanup 3 4 1. Nordoxepin 2. Doxepin 3. Nortriptyline 4. Amitryptyline 99.4 ± 4.3% 102.1 ± 1.8% 95.6 ± 2.7% 101.3 ± 3.2% 6 SPE: Discovery DSC-MCAX, 100mg/3mL, HPLC: Discovery C8, 15cm x 2.1mm ID, 5µm, Mobile Phase: 10mM ammonium acetate, ph 4.5:MeCN (45:55), Flow Rate: 0.42mL/mi,; Temp.: 40ºC, Det.: 210nm, UV, Inj. Vol.: 10µL
Case 2: Extraction of 2-Aminopyridine & Piroxicam from Human Urine Spiked Urine Sample Without SPE Cleanup 1 Spiked Urine Sample After DSC-MCAX 2 1. 2-aminopyridine (4.0µg/mL spike) % Abs Recovery + RSD (n=4) Discovery DSC- MCAX SPE Vendor A Mixed- Cation SPE 2. Piroxicam (10µg/mL spike) 2-aminopyridine 102 ± 3.5% 30 ± 52.5% Piroxicam 101 ± 1.2% 98 ± 3.2% Vendor B Mixed- Cation SPE 36 ± 24.2% 83 ± 4.3% SPE: Discovery DSC-MCAX, 100mg/3mL, Vendor A & B Mixed-Cation SPE, 100mg/3mL, HPLC: Discovery HS F5, 15cm x 4.6mm ID, 5µm, Mobile Phase: 10mM potassium phosphate, ph 6:MeCN (85:15), Flow Rate: 2mL/min, Temp.: 25 C, Det.: 220nm, UV, Inj. Vol.: 10µL
Case 3: Importance of Ion-Exchange Capacity on Recovery Drug DSC-MCAX Vendor A Vendor B Buprenorphine 98.4±1.2 91.3±8.2 85.7±6.4 Codeine-6-glucuronide 89.3±1.1 88.4±10.8 72.3±14.2 Ecgonine Methyl Ester 89.4±2.8 84.2±13.3 76.8±15.5 Isoxsuprine 97.4±1.8 92.3±6.9 84.7±7.7 Levallorpham 98.3±1.9 98.2±2.8 92.6±6.7 6-Monoacetyl Morphine 98.4±1.2 91.3±8.2 85.7±6.4 Morphine 94.5±3.9 84.3±11.2 68.7±15.4 3-Pyridylacetontrile 94.6±4.1 51.4±21.2 33.7±28.5 Vendor A has an ion-exchange capacity of 2.4mg atrazine per gram and the value for Vendor B is 1.2mg/g. Lower recoveries of these compounds are assigned to the partial losses in the previous washing step. The present study evidences that an ion-exchange capacity over 3.6mg atrazine per gram is essential. DSC-MCAX has the value between 3.6mg/g and 4.8mg/g.
Generic Extraction Protocol 2 Weakly Basic, Polar Basic, Weak Zwitterionic, or Polar Zwitterionic Drug Generic Extraction Protocol (for 100mg/1mL or 100mg/3mL SPE tube) When a target may have (even a minor but detectable) leakage during loading at neutral ph, the second protocol should be used: 1. Sample Pre-Treatment: Dilute urine (plasma) sample (1mL) with 10mM potassium phosphate buffer (ph3.0) or 10mM acetic acid buffer (ph3.0) (1mL). 2. SPE Tube Conditioning: Solvate the tube with 1mL methanol 3. SPE Tube Equilibrium: Equilibrium the tube with 10mM ph3.0 potassium phosphate buffer or 10mM acetic acid buffer (1mL) 4. Sample Loading: Load pre-treated urine or plasma (1mL) at a flow rate of 1mL/min 5. Interference Elution: Elute interference with 1mL 10mM ph3.0 phosphate buffer (or acetic acid buffer) and methanol (1mL). 6. Analyte Elution: Elute analytes with 5% ammonium hydroxide in methanol
Case 4: Vendor Comparison - Extraction of Cytosine Using Mixed-Mode Exchange SPE Discovery DSC-MCAX SPE Absolute Recovery: 99.2 ± 4.8% (n=3) Vendor A Mixed-Cation SPE Average Recovery: 1.8% Vendor B Mixed-Cation SPE Average Recovery: 4.2-44.0% SPE: Discovery DSC-MCAX, 100mg/3mL, Vendor A & B Mixed-Cation SPE: 100mg/3mL, HPLC: Discovery HS F5, 15cm x 4.6mm ID, 5µm, Mobile Phase: 10mM potassium phosphate, ph 3, Flow Rate: 1mL/min, Temp.: Ambient, Det.: 280nm, UV, Inj. Vol.: 10µL
Absolute Recoveries of Polar Zwitterions Compounds by DSC-MCAX Drug DSC-MCAX Alprazolam 98.7 ± 1.9 Brombuterol 99.7±1.1 Buprenorphine 102.4±4.6 Clenbuterol 104.8±3.3 Diazepam 96.3±2.6 Ecgonine 92.9±3.8 Estazolam 101.8±2.8 Mabuterol 89.3±4.2 Mapenterol 94.5±2.7 Methaqualone 99.5±2.2 Nordazepam 94.8±3.2 Oxazepam 92.8±3.8 Oxytetracycline 87.9±3.3 S(+)-Salbutamol 91.4±2.5 Terbutaline 90.4±4.2 Comments Zwitterions compounds such as benzodiazepines have been considered as neutral compounds, and thus are frequently distributed into two fractions, i.e. the methanol fraction and the final fraction when the mixed-mode SPE cartridges from the current vendors are used. However, at ph3.0, the compounds are positively charged and when DSC-MCAX is used, they are fully eluted into the final fraction with high recoveries.
Generic Extraction Protocol 3 Systematic Fractionation of Neutral and Acidic Compounds from Basic and Zwitterionic Compounds The protocol was developed by Takeda et al. (J. Chromatogr. B. 2001, 758, 235) 1g/6mL SPE tube was used via the protocol described below: 1. Equilibrate the SPE tube by 6mL of methanol 2. Condition the SPE tube by 6mL of 10mM acetic acid (ph3) 3. The ph of the biological fluid sample should be adjusted to ph 3.5 or below in order to keep acidic compounds by the SPE tube via reversedphase mechanism. Under the low ph condition, both basic and zwitterionic compounds were kept mainly by ion-exchange mechanism. 1mL or more of the sample was loaded by the SPE tube. 4. Wash the SPE by 6mL of 10mM acetic acid (ph3) and this process should last at least ten minutes 5. Dry the SPE tube by 6mL chloroform. The moisture in the SPE tube is removed. 6. Recover acid and neutral compounds by 6mL acetone:chloroform (20:80). 7. Recover the basic and zwitterionic fraction by 6mL 5% ammonium hydroxide in methanol.
Case 5: Systematic Fractionation and Analysis of Acidic and Basic Drugs by DSC-MCAX Sample Prior to DSC-MCAX SPE mau 1 3 5 2 4 DSC-MCAX Fraction 2: Basic Compounds 3 5 mau 0 6 0 6 DSC-MCAX Fraction 1: Acidic Compounds mau 0 10 6 1 2 4 Compound 1. Secobarbital 2. Keprofen 3. Nortriptyline 4. Naproxen 5. Amitryptyline % Abs Recovery ± RSD (n=3) 105.8±2.1 101.7±1.3 100.3 ±0.5 101.5±0.8 103.3±1.7
Case 6: Fractionation of Benzoic and p-nitrobenzoic Acid Using DSC-MCAX Sample Prior to DSC-MCAX SPE 1 3 DSC-MCAX Fraction 1: 3 Acidic Compounds 2 2 1 DSC-MCAX Fraction 2: Only o-aminobenzoic acid 1. o-aminobenzoic acid 2. Benzoic acid 3. p-nitrobenzoic acid SPE: Discovery DSC-MCAX, 300mg/3mL, HPLC: Discovery C18, 15cm x 4.6mm ID, 5µm, Mobile Phase: 0.1% TFA:MeOH (60:40), Flow Rate: 2mL/min, Temp.: Ambient, Det.: 254nm, UV, Inj. Vol.: 10µL
Case 6. Vendor Comparison Acidic fraction by Vendor A: 22% Acidic fraction by Vendor B: 92% o-aminobenzoic acid eluted into the o-aminobenzoic acid eluted into the fraction. fraction. mau 0 20 1 Basic fraction: 77% o-aminobenzoic acid 1 mau 6 8 10 mau 0 20 1 Basic fraction: only 8% o-aminobenzoic acid in the fraction 1 mau 0.0 0.2 0.4 0.6
Comparison Between Silica- and Polymer-Based Mixed-Mode SPE Silica-Based Mixed-Mode: 1. Biological samples loaded at ph 3-6. At higher ph, the final basic and zwitterions fraction has cleanest background. Reducing ion-suppression for LC-MS quantitation is critical. 2. A number of successful applications for systematic fractionation of acid, neutral and basic compounds. 3. Adequate hydrophobicity for non-polar compounds. Polymer-Based Mixed-Mode: 1. Biological samples loaded at ph 1. A centrifuge step may be required to remove sediments. Extract background is frequently not as clean as that from silica-based mixed-mode SPE. 2. Few applications on file for systematical fractionation of acid, neutral and basic compounds. 3. Many basic compounds such as morphine were lost during the methanol wash step due to the inadequate hydrophobicity.
C8+SCX Mixed-Mode SPE: Vendor Comparison Current C8+SCX Mixed-Mode SPE: 1. The application range is severely limited to non-polar basic compounds. 2. Greater risk to lose polar compounds and/or basic compounds with lower pka. 3. Zwitterions compounds may be partially lost during sample load and wash steps resulting in elution distribution across multiple fractions. Discovery DSC-MCAX: 1. Optimized ion-exchange capacity to extract and recover a wide range of basic compounds (from weak to strong, polar to hydrophobic). 2. Reduced risk of prematurely eluting polar basic, weak basic and polar zwitterions compounds. 3. Zwitterionic compounds will be retained at ph 3.0, and can be recovered with basified methanol during final elution.
Conclusion Three generic extraction protocols were described in this report. The first two methods discussed the extraction of basic compounds at two ph levels. By loading samples at the appropriate ph, strong ionic and hydrophobic interactions can occur during the extraction process. As a result, strong wash solvents (e.g., methanol) can be used to elute co-retained interferences thereby drastically improving selectivity final elution with basified methanol. The third method illustrated the use of mixed-cation phases to fractionate basic and zwitterionic compounds from neutral and acidic compounds. Each application describes the importance of ion-exchange capacity on the recovery of polar basic and polar zwitterionic compounds. Unlike most commercially available mixed-cation phases, Discovery DSC-MCAX offers the ion-exchange capacity necessary to retain and adsorb all basic and zwitterionic compounds until final elution. For fractionation applications, poor ion-exchange capacity can result in partial distribution of polar basic and zwitterionic compounds across multiple fractions.