Evaporative crosstalk is contamination that occurs during evaporation of the elution solvent after SLE or SPE extraction. Volatile analytes evaporate, then redistribute in adjacent wells in a 96-well plate. This can generate false positive results and require re-extraction of patient specimens. Addition of HCl in methanol (MeOH) to produce the salt of volatile compounds like sympathomimetic amines is used to control crosstalk. The type of evaporator, conditions and solvents used can all contribute to crosstalk.
Crosstalk can still persist and cause contamination of negative samples even with the addition of HCl in MeOH. The Biotage® ACT Adapter reduces crosstalk during evaporation. Reducing crosstalk improves integrity of results and reduces the number of clinical specimens that need to be re-extracted.
We have customers that have reported urine concentrations of amphetamine and methamphetamine in excess of 100,000 ng/mL. We conducted multiple experiments in our Charlotte applications lab to determine how to control evaporative crosstalk in these high concentration samples using the ACT Plate Adapter and HCl in MeOH. Eight drugs and metabolites were evaluated: amphetamine, methamphetamine, MDA, MDMA, MDEA, benzoylecgonine, morphine and hydromorphone. An extraction blank and urine calibrator populated the first column of each plate. Three spiked urine specimens at concentrations between 50,000 and 100,000 ng/mL were placed in different areas of the plate.
Initial experiments showed no evaporative crosstalk for benzoylecgonine, morphine or hydromorphone with or without the ACT Plate Adapter and the addition of 10 µL of 1 mM HCl in methanol. Some crosstalk was detected in drug free urine samples adjacent to a spiked sample for MDA, MDEA and MDMA but was reduced to <5 ng/mL when the ACT Plate Adapter was used. Evaporative crosstalk with concentrations in drug free wells between 2 and 200 ng/mL were observed for methamphetamine and amphetamine without the ACT Plate Adapter (Figure 1). This was reduced to 1 to 100 ng/mL when the ACT Plate Adapter was used (Figure 2). This was still too high for many clinical and forensic assays. Further work focused on reducing crosstalk for methamphetamine and amphetamine only. The rest of the plate was populated with samples of drug free urine. Experiments were done with and without the ACT Plate Adapter, and with different concentrations of HCl in MeOH. 150 µL of urine was treated with 165 µL of a master mix designed to be consistent with reagents added for enzyme hydrolysis: pH7 phosphate buffer, methanol (to mimic addition of internal standard solution) and water. Samples were not hydrolyzed. The samples were then treated with 0.1% ammonium hydroxide, and extracted using a 400 µL ISOLUTE SLE+ plate, eluting with 2 x 600 µL of 90:10 dichloromethane:2-propanol. The elution solvent was evaporated using a SPE Dry and reconstituted and analyzed following the LC-MS/MS method in the Biotage® Urine White Paper.
Figure 1. Crosstalk without the ACT Plate Adapter and 10 µL of 1 mM HCl in methanol. Yellow are spiked samples. Pink are concentrations >20 ng/mL in negative samples. Figures 4 and 5 only had methamphetamine and amphetamine spiked in wells C4 and F7.
Figure 2. Crosstalk using the ACT Plate Adapter and 10 µL of 1 mM HCl in methanol. Yellow are spiked samples. Pink are concentrations >20 ng/mL in negative samples. Figures 4 and 5 only had methamphetamine and amphetamine spiked in wells C4 and F7.
Next, the concentration of HCl in MeOH was increased. Extractions were performed and either 10 µL of 0.25% HCl in MeOH or 10 µL of 0.5% HCl in MeOH (Figures 3 and 4) were added to separate extracted plates prior to evaporation. Some crosstalk was still observed at both concentrations, but this was reduced to <30 ng/mL for both analytes with 10 µL of 0.5% HCl in MeOH and the ACT Plate Adapter.
Figure 3. Crosstalk using the ACT Plate Adapter and 10 µL of 0.25% HCl in methanol. Yellow are spiked samples. Pink are concentrations >20 ng/mL in negative samples.
Figure 4. Crosstalk using the ACT Plate Adapter and 10 µL of 0.5% HCl in methanol. Yellow are spiked samples. Pink are concentrations >20 ng/mL in negative samples.
The ACT Plate Adapter reduced crosstalk for MDA, MDEA and MDMA, methamphetamine and amphetamine, but the correct concentration of HCl in MeOH is still required to “salt out” the methamphetamine and amphetamine present in very high concentration samples. The SAMHSA (Substance Abuse and Mental Health Services Administration) confirmation cutoff for methamphetamine and amphetamine in urine is 250 ng/mL. The combination of 10 µL of 0.5% HCl in MeOH and the use of the ACT Plate Adapter reduced crosstalk to <30 ng/mL. This should be acceptable for most clinical and SAMHSA drug assays using urine specimen volumes of 150 µL or less. Elution volume also plays a role in reducing crosstalk. Reducing the urine volume (and hence sample volume) and using a 200 µL ISOLUTE® SLE+ plate with half the elution volume (2 x 300 µL) reduces the incidence of evaporative crosstalk even further.
To learn more, read our technical note “Avoiding Cross Talk In 96-well Based Sample Preparation”.