LC/MS/MS Analysis of Diuretics in Urine Proper Column Choice Takes Matrix out of the Equation

• Ultra II™ Biphenyl columns resolve compounds that coelute on phenyl hexyl columns.
• Improve quantitation through resolution of compounds from isobaric matrix interferences.
• Fast, 8-minute analysis, including re-equilibration.

The most common method for the detection of performance enhancing drugs, also known as doping agents, is urine analysis. Diuretics are often used in an effort to mask the presence of performance enhancing drugs since they act to dilute urine. Because of this, the use of diuretics has been banned by the World Anti-Doping Agency (WADA). Most common diuretics are included in the WADA list of banned substances, including carbonic anhydrase inhibitors, potassium-sparing diuretics, loop diuretics, and thiazides (Figure 1). Due to the high degree of functionalization of these molecules, many of them are hydrophilic, which can make them very difficult to retain using conventional C18 columns. Phenyl columns are a good alternative, as they generally have better hydrophilic retention; however, not all phenyl columns produce adequate results.

Figure 1: Structures of diuretics used to mask doping agents.

Using an Ultra II™ Biphenyl column in combination with LC/MS/MS provides fast, reliable resolution of target diuretics from matrix interferences. Although the use of LC/MS/MS often can eliminate the need for chromatographic separation, this is not the case when isobaric interferences—matrix components with MS/MS transitions identical to those of target compounds—are present. Despite the power of modern LC/MS/MS instrumentation, isobaric matrix interferences often complicate analyses involving biological samples and using a column that produces adequate retention is critical for accurate quantitation. In urine, a very intense region of interference occurs at the beginning of the analytical run, and an easy way to reduce the risk of interference is to retain target compounds with a minimum k' value of between 1 and 2.

Ultra II™ Biphenyl columns can retain hydrophilic compounds longer than other phenyl-based stationary phases, due to the unique selectivity of the Biphenyl ligand for highly-functionalized aromatic compounds. As shown in Figure 2, using an Ultra II™ Biphenyl column ensures complete separation of target analytes (k' = 5). In contrast, matrix interference occurs on a Gemini® C6-Phenyl (phenyl hexyl) column (k' = 0.6), preventing accurate quantitation of amiloride using the 230/60 m/z transition. Note that the concentration of analytes in matrix was relatively high; as the concentration is lowered, matrix effects become even more problematic.

Figure 2: Higher retention on Ultra II™ Biphenyl columns allows quantitation of diuretics that coelute with matrix on other phenyl phases.

A. Ultra II™ Biphenyl Column: Ultra II™ Biphenyl (cat.# RE9609352) Dimensions: 50mm x 2.1mm ID Particle Size: 3µm Pore Size: 100Å Temp.: 40°C B. Phenomenex® Gemini® C6 Phenyl Hexyl Column: Gemini® C6-Phenyl (phenyl hexyl) Dimensions: 50mm x 2.0mm ID Particle Size: 3µm Pore Size: 110Å Temp.: 40°C Sample: Conc.: 50ng/mL diuretics in urine, diluted 10x in mobile phase Inj. vol.: 5µL Mobile phase: A: 0.1% formic acid in water B: 0.1% formic acid in methanol Time (min.) %B 0 10 6.00 100 6.10 10 8.00 10 Flow: 0.5mL/min. Detector: Det.: Applied Biosystems/MDS Sciex LC/MS/MS Model #: API 5000 Ion source: TurboIonSpray® Ion mode: ESI+ Ion spray voltage: 5.5kV Curtain gas: 25psi (172.4 kPa) Gas 1: 60psi (413.7 kPa) Gas 2: 40psi (275.8 kPa) Source temp.: 550°C Mode: MRM Dwell time: 50ms Instrument: Shimadzu Prominence UFLCXR

Although early eluting compounds are well retained on the Ultra II™ Biphenyl column, fast analytical run times can still be obtained. The Biphenyl column's optimized selectivity allows for resolution of all target diuretics in just 8 minutes, including re-equilibration time. Figure 3 shows the analysis of all of the compounds of interest, some of which were analyzed in a separate run using negative ionization.

Figure 3: Fast, selective analysis of 500ng/mL diuretics in urine on an Ultra II™ Biphenyl column. Excellent retention is achieved for all compounds.

A. Positive ionization MRM1 (Q1/Q3) MRM2 (Q1/Q3) MRM3 (Q1/Q3) 1. Amiloride 230/171 230/116 230/60 2. Triamterene 254/237 254/104 254/141 3. Indapamine 366/132 366/91 366/117 4. Ethacrynic acid 303/179 303/194 303/257 5. Bumetanide 365/240 365/156 365/284 B. Negative ionization MRM1 Q1/Q3 MRM2 Q1/Q2 MRM3 Q1/Q2 1. Acetazolamide 221/83 221/80 221/58 2. Chlorothiazide 294/214 294/179 294/215 3. Hydrochlorothiazide 296/78 296/269 296/205 4. Bendroflumethiazide 420/289 420/328 420/197 5. Furosemide 329/285 329/205 329/78 Column: Ultra II™ Biphenyl (cat.# RE9609352) Dimensions: 50mm x 2.1mm ID Particle Size: 3µm Pore Size: 100Å Temp.: 40°C Sample: Conc.: 500ng/mL diuretics in urine, diluted 10x in mobile phase Inj. vol.: 5µL Mobile phase: A: 0.1% formic acid in water B: 0.1% formic acid in methanol Time (min.) Flow (mL/min.) %A %B 0 0.5 90 10 6.00 0.5 0 100 6.1 0.5 90 10 8.00 0.5 90 10 Detector: Det.: Applied Biosystems/MDS Sciex LC/MS/MS Model #: API 5000 Ion source: TurboIonSpray® Curtain gas: 25psi (172.4 kPa) Gas 1: 60psi (413.7 kPa) Gas 2: 40psi (275.8 kPa) Source temp.: 550°C Mode: MRM Dwell time: 50ms Instrument: Shimadzu Prominence UFLCXR

Although chromatographic resolution is not always required for LC/MS/MS analyses, it is necessary when isobaric interferences are present, as often occurs in the analysis of biological matrices. Biphenyl columns are highly retentive, providing a valuable tool for the analysis of biological samples. In this analysis of diuretics in urine, only the Ultra II™ Biphenyl column provided adequate resolution of target diuretics from matrix components.