Quantitation of vitamin K1 in serum using online SPE-LC-MS/MS and the challenges of working with vitamin K.

Vitamin K1 (phylloquinone) is one of the vitamin Ks. Several studies have previously investigated the role of vitamin K1 status in respect to disease, but without consistent results. Since vitamin K deficiency has been associated with different disease states it is important to develop a biochemical analysis with sufficient sensitivity and a low limit of quantitation (LOQ). The vitamin Ks are very fat-soluble. This non-polar nature has given rise to several challenges during the method development, because vitamin K1 sticks to materials used during the process and is lost during evaporation. We found that reducing the sample preparation as much as possible offline, instead using online SPE-LC-MS/MS improves recovery and gives satisfactory chromatograms. An Protein BEH C4 column, 300 Š(50 × 2.1 mm, 1.7 µm particle size) was used as trap column and a Phenyl-Hexyl-LC-column, 100 Š(100 × 2.1 mm, 2.6 µm particle size) was used as analytical column. The mobile phases consisted of 30 µmol/L NH4F in water and 30 µmol/L NH4F in MeOH. A triple quadrupole tandem mass spectrometer with atmospheric pressure chemical ionization (APCI) ion source, positive ion mode, was used to perform the mass spectrometric measurements. The method is simple, highly sensitive and fast. The method was validated for vitamin K1 with good analytical performance. With a LOQ of 0.05 nmol/L it is to our knowledge the vitamin K1 method with lowest LOQ reported to date in the literature. It can easily be automated and applied in a routine diagnostic laboratory. Blood collection tubes with different additives were tested and showed no difference. Stability of vitamin K1 in serum was tested at different temperatures (-20 °C, 4 °C and in light and dark at 20 °C over a period of 30 days) and showed that vitamin K1 is light sensitive in serum even after only one day.

Utility of testing for monoclonal bands in serum of patients with suspected osteoporosis: retrospective, cross sectional study.

OBJECTIVE: To determine whether measuring monoclonal bands (M component) in serum should be part of the investigation of patients referred to osteoporosis clinics. DESIGN: Retrospective, cross sectional, observational study. SETTING: Referral centre for osteoporosis in a university hospital, Denmark. PARTICIPANTS: 799 people (685 women) aged 19 to 94 years newly referred with suspected osteoporosis. MAIN OUTCOME MEASURES: Proportion of patients fulfilling the Nordic Myeloma Study Group definition for target condition and proportion of patients with other important haematological conditions. RESULTS: 4.9% (18 of 366) of patients with osteoporosis and 2.2% (9 of 408) of patients without osteoporosis had M component in serum (chi2 = 3.66, P = 0.04). Multiple myeloma was diagnosed in three patients with osteoporosis (absolute risk 0.8%, 95% confidence interval 0.11% to 1.7%). The relative risk of multiple myeloma in patients presenting with osteoporosis was 75 (10 to 160). As a diagnostic test for multiple myeloma in patients with osteoporosis, M component in serum had a specificity of 95.0% and a positive predictive value of 17.6%. 122 blood electrophoreses were carried out for each case of multiple myeloma diagnosed. All patients with multiple myeloma had a history of fragility fractures. If lymphoma was included as a target condition, the specificity increased to 95.3% and the positive predictive value increased to 23.5%. Monoclonal gammopathy of undetermined significance was diagnosed in 13 (3.6%) participants with osteoporosis and in eight (2.0%) participants with normal bone mineral density or osteopenia. CONCLUSIONS: Patients presenting with osteoporosis should be tested for M component in serum, as 1 in 20 patients with newly diagnosed osteoporosis had multiple myeloma or monoclonal gammopathy of undetermined significance.