Calibrator
Vol. of 200 ng/mL stock added (μL)
Vol. of MeOH added (μL)
Urine added (μL)
Total vol. (mL)
Approx. final conc. (pg/mL)a
1
0
750
99,250
100
0
2
10
740
99,250
100
20
3
125
625
99,250
100
250
4
250
500
99,250
100
500
5
500
250
99,250
100
1000
6
750
0
99,250
100
1500
2.
Check the new lot of calibrators by verifying five unknown patient specimen with the current lot of calibrators. The agreement between the two calculated concentrations must be within 15 %.
3.
Controls: Prepare high and normal control materials by pooling previously analyzed samples. The high-quality control material may be prepared by the addition of 25(OH)D2 and 25(OH)D3 to pooled normal urine. The controls are stable for 4 months at −20 °C.
4.
Establish a range for the new lot of controls by collecting data points over 20 consecutive runs and establish the mean and standard deviation.
2.5 Analytical Equipment and Supplies
1.
Prominence LC-20 (Shimadzu) coupled to an Applied Biosystems API 5000 triple quadrupole mass spectrometer (AB Sciex).
2.
HPLC Column: Gemini C18, 50 × 2.0 mm i.d., 3 μm particle (Phenomenex).
3.
Centrifuge tubes and National Scientific 2 mL amber glass vials with inserts and pre-slit caps or equivalent.
4.
TurboVap LV evaporator (Biotage).
5.
CEREX 48 positive pressure manifold (SPEware).
6.
Trace-N SPE cartridge, 3CC (SPEware).
2.6 Instrument Operating Conditions
1.
High-performance liquid chromatography (HPLC ): A Schimadzu Prominence LC-20 system consisted of an autosampler, column oven, and two micro pumps. Chromatographic separations of 25(OH)D2 and 25(OH)D3 as well as the internal standard were achieved using a 50 × 2.0 mm i.d., 3 μm particle size Gemini C18 column maintained at 45 °C. mobile phase A consisted of 1 % methanol in water and 2.5 mM ammonium formate, and mobile phase B consisted of methanol and 2.5 mM ammonium formate. The HPLC method is described in Table 2. The injection volume is 50 μL (see Note 4 ), with a syringe wash volume of 250 μL using the syringe wash solvent. The autosampler performs one wash pre-injection and three washes post-injection.
Table 2
HPLC method
Step | Total time (Min) | Flow rate (μL/min) | %A | %B |
|---|---|---|---|---|
1 | 1.0 | 250 | 25 | 75 |
2 | 6.0 | 250 | 5 | 95 |
3 | 6.1 | 300 | 5 | 95 |
4 | 6.5 | 300 | 5 | 95 |
5 | 6.6 | 300 | 25 | 75 |
6 | 7.5 | 300 | 25 | 75 |
2.
Tandem mass spectrometry : Mass spectrometric detection was performed using an Applied Biosystems API 5000 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in a positive ion mode. Multiple reaction monitoring (MRM) was selected for detection of 25(OH)D2 and 25(OH)D3 and their respective internal standards with a dwell time of 250 ms (Fig. 1). As shown in Table 3, two mass transitions were monitored for each analyte. The tune parameters used for data acquisition were: source temperature of 300 °C; collision activation dissociation (CAD) gas value of 3; curtain gas of 20 psi; nebulizer gas of 35 psi; heating gas of 30 psi; and a spray voltage of 4800 V. The DP, EP, CXP, CEM, and DF were 52, 13, 24, 2200, and 100 V, respectively. Nitrogen (99.995 % purity) was used as the desolvation and collision gas. The MRM acquisition method was run in unit resolution (0.7 amu) in both Q1 and Q3.
