5 Minute Analysis of Vitamin D in Serum by LC/MS/MS

• High sensitivity improves low level accuracy in matrix
• 5 minute analysis time speeds up sample throughput.

Excellent selectivity for vitamin D minimises matrix interference

Monitoring of vitamin D levels in patients is important for the prevention and control of disease. Vitamin D, specifically 25-hydroxy vitamin D, plays a critical role in controlling calcium and phosphate levels in the body. If these levels are not adequately controlled, bone conditions such as rickets in children or osteoporosis in adults may occur. 25-hydroxy vitamin D is a hydrophobic, fat soluble vitamin that is absorbed like a fat in the intestines. It is commonly used to diagnose conditions that interfere with fat absorption, such as Crohn's disease. Since vitamin D analysis is one of the most commonly run procedures in clinical labs, high throughput, high sensitivity analytical methods are desirable. Conventional techniques for vitamin D analysis, based on immunoassay or LC/UV, often lack adequate sensitivity, specificity, and speed; thus, interest in LC/MS/MS methods is growing. Here we establish conditions for routine vitamin D testing by LC/MS/MS which result in highly symmetric peaks that elute in just 5 minutes.

25-hydroxy vitamin D appears in several forms, but vitamin D2 and vitamin D3 are the most commonly analyzed. These forms are very similar and differ only in one methyl group and a double bond (Figure 1). Because 25-hydroxy vitamin D is a hydrophobic species, this compound is extremely amenable to reverse phase liquid chromatography (RPLC). While conventional C18 columns are commonly used in RPLC, for this analysis we selected an Ultra Aqueous C18 column instead. This phase is more retentive than a C18, which helps separate the vitamin D species from less retained matrix components. LC/MS/MS analysis using an Ultra Aqueous C18 column resulted in excellent peak shape, which contributes to enhanced sensitivity (Figure 2). To evaluate retention, human serum samples were extracted in acetonitrile and analyzed. Both vitamin D analytes were well-separated from matrix interferences (Figure 3).

Analyzing vitamin D by LC/MS/MS using an Ultra Aqueous C18 column is an ideal method for high-throughput clinical labs interested in accurate low-level detection and fast analysis times. Excellent peak shape and MS sensitivity result in faster, more accurate analysis of clinical samples.

Figure 1: Structures of vitamins D2 and D3.

Figure 2: Ultra Aqueous C18 columns provide outstanding peak symmetry for vitamin D, improving accuracy at low concentrations.

Compound Q1 Q3 Declustering potential (V) Ret. Time (min.) d6-25-OH D3 (IS) 389.3 211.2 68 3.00 25-OH D3 383.3 211.2 68 3.00 25-OH D2 395.3 229.2 55 3.04 25-OH D3 383.3 229.2 68 3.00 25-OH D2 395.3 269.2 55 3.04 25-OH D2 395.3 119.0 55 3.04 Column: Ultra Aqueous C18 Cat. #: 9178352 Dimensions: 50mm x 2.1mm Particle size: 3µm Pore size: 100Å Sample: Inj.: 20µL Conc.: 20ng/mL vitamin D standard Conditions: Instrument: Shimadzu Prominence® UFLCXR Mobile phase: A: 0.1% formic acid in water B: 0.1% formic acid in methanol Time (min.) %B 0.0 50 2.5 100 3.5 100 3.6 50 5.0 50 Flow: 700µL/min. Temp.: 40°C Det.: Applied Biosystems 3200 QTRAP® LC/MS/MS System Ion Source: TurboIonSpray®, APCI+ Mode: MRM Dwell time: 100msec. LC_CF0491

Figure 3: Excellent results for vitamin D in patient serum can be obtained in just 5 minutes.

Compound Q1 Q3 Declustering potential (V) Ret. Time (min.) d6-25-OH D3 (IS) 389.3 211.2 68 3.00 25-OH D3 383.3 211.2 68 3.00 25-OH D2 395.3 229.2 55 3.04 25-OH D3 383.3 229.2 68 3.00 25-OH D2 395.3 269.2 55 3.04 25-OH D2 395.3 119.0 55 3.04 Column: Ultra Aqueous C18 Cat. #: 9178352 Dimensions: 50mm x 2.1mm Particle size: 3µm Pore size: 100Å Sample: Inj.: 20µL Conc.: extracted serum sample Conditions: Instrument: Shimadzu Prominence® UFLCXR Mobile phase: A: 0.1% formic acid in water B: 0.1% formic acid in methanol Time (min.) %B 0.0 50 2.5 100 3.5 100 3.6 50 5.0 50 Flow: 700µL/min. Temp.: 40°C Det.: Applied Biosystems 3200 QTRAP® LC/MS/MS System Ion Source: TurboIonSpray®, APCI+ Mode: MRM Dwell time: 100msec. LC_CF0492