Table 3.
Mass spectrometric methods applicable to clinical laboratories.
| Ref | Sample volume, extraction procedure, chromatographic procedure, ionization, mode of monitoring | Internal standards, analyte measured, acquisition settings, m/z | Recoverya | LOQ nmol/L, LOD nmol/L | Precision (CV) Intra-assay Inter-assay |
|---|---|---|---|---|---|
| Higashi et al. [80] | Plasma 20 μl, PP: MeCN, extraction: LLE (AcOEt), derivatisation (DMEQ-TAD) HPLC: J׳sphere ODS H-80 MeCN/H2O (3/2 v/v) TMS: APCI+ SIM | In-house IS: 25OHD4: 760.1, 25OHD3: 746.1, 25OHD2: 758.1 | 25OHD3: 98.8–109.8% (12.5 nmol/L), 25OHD2: 101.1–104.2% (12.5 nmol/L) | 25OHD3: 7.5, 25OHD2: 7.5, 25OHD3: 1.3 | 25OHD3: 3.24% at 21.9 nmol/L, 25OHD2: 3.17% at 12.5 nmol/L |
| Higashi et al. [81] | Plasma 20 μl, PP: MeCN, extraction: LLE (AcOEt), derivatisation (NPTAD) HPLC: J׳sphere ODS H-80 MeOH/H2O (7/1 v/v) TMS: APCI− SIM | In-house IS: 25OHD4: 634.2, 25OHD3: 620.2 | Analytical recovery: NR | 25OHD3: 7.5, 25OHD3: 1.3 | 25OHD3: 8.2% at 7.5 nmol/L |
| Vogeser et al. [82] | Serum 200 μl, NaOH, PP: MeCN, extraction: on-line SPE: Oasis HLB® HPLC: LiCrospher® 100 RP-18 MeOH/NH4Ac:0.5mM (90/10 v/v) TMS: ESI+ | In-house IS: 2H3, 13C1-25OHD3: 405>159, 25OHD3: 401>159 | 25OHD3: 91±1.6% IS (325 nmol/L), injected into TMS/IS, extracted+TMS | NR | 25OHD3: 12% at 14.5 nmol/L |
| Tsugawa et al. [83] | Serum 100 μl, PP: MeOH, extraction: SPE: Bond-Elute C18® HPLC: CapCell PAK C-18 UG120® MeOH/H2O (95/5 v/v) TMS: APCI+ MRM | In-house IS: 2H6-25OHD3: 407>263, 25OHD3: 401>257, 25OHD2: 413>255 | 25OHD3: 103.8% (50 nmol/L), 25OHD2: 98.8% (7.5 nmol/L) | 25OHD3: 2.5, 25OHD2: 2.5 | 25OHD3: 5.7% at 50 nmol/L, 25OHD2: 4.5% at 7.5 nmol/L, 25OHD3: 2.5% at 47.5 nmol/L, 25OHD2: 5.1% at 8.0 nmol/L |
| Maunsell et al. [84] | Serum 100 μl, PP: MeOH:Propanol (80:20 v/v), Extraction: LLE: n-Hexane HPLC: BDS C8® ThermoHypersil MeOH>H2O+0.05% CHO2H Gradient TMS: ESI+ MRM | In-house IS: 2H6-25OHD3: 407.2>389.4, 25OHD3: 401.8>383.5, 25OHD2: 413.5>395.4 | 25OHD3: 91–110% at 128–256 nmol/L, 25OHD2: 94–108% at 158–317 nmol/L | 25OHD3: <4.0, 25OHD2: <5.0 | 25OHD3: 6.2% at 16 nmol/L, 25OHD3: 5.1% at 55 nmol/L, 25OHD2: 9.5% at 52 nmol/L |
| Chen et al. [85] | Serum 200 μl, PP: MeCN Extraction: SPE: Oasis HLB® MeOH/H2O (30/70 v/v); MeCN/MeOH (50/50 v/v), HPLC: SupelCosil®LC-18-DB EtOH:H2O (83:17 v/v) TMS: APCI+ MRM | Commercial IS: 2H6-25OHD3: 407.7>389.7, 25OHD3: 401.4>383.4, 25OHD2: 413.4>395.4 | 25OHD3: 99±2% at 34.2–132.8 nmol/L, 25OHD2: 95±0.8% at 32.2–115.5 nmol/L | 25OHD3: 4.0, 25OHD2: 15.5, 25OHD3: 1.2, 25OHD2: 4.6 | 25OHD3: 6.2% at 34 nmol/L, 25OHD2: 8.7% at 23 nmol/L, 25OHD3: 11% at 34 nmol/L, 25OHD2: 16% at 23 nmol/L |
| Bunch et al. [86] | Serum 100 μl, PP: MeOH Extraction: OLTFE HPLC: Hypersil Gold aQ® MeOH/H2O (95/5 v/v) TMS: APCI+ MRM | In-house IS 2H6-25OHD3: 407.2>389.4, 25OHD2: 413.5>395.4, 25OHD3: 401.8>383.5 | 25OHD3: 3.0, 25OHD2: 4.6 | ||
| Hojskov et al. [87] | Serum 100 μl, PP: MeCN, extraction: automated LLE: 96-well Isolute HM-N plate®/diatomaceous earth; Heptane HPLC: Synergi MAX-RP® MeOH/2.0 mM NH4Ac (85/15 v/v) TMS: APCI+ MRM | Commercial IS 2H6-25OHD3: 407.4>371.4 25OHD3: 401.4>365.2, 25OHD2: 413.4>395.4 | NR | 25OHD3: <10, 25OHD2: <10 | 25OHD3: 9.4% at 32 nmol/L, 25OHD2: 8.6% at23.4 nmol/L |
| Hermann et al. [88] | Serum 100 μl, PP: MeCN HPLC: Supelcosil LC-8® H2O>MeOH>H2O/MeOH (98/2 v/v)>Toluene APPI+ MRM | Commercial IS 2H6-25OHD3: 389>371, 2H6-25OHD2 401>383, 25OHD3: 395>377, 25OHD2: 413.5>395.4 | 108–113%, expressed as total 25OHD added (45–90 nmol/L) | 25OHD3: 1.3, 25OHD2: 1.3 | 25OHD: 5.7% at 17 nmol/L, 25OHD: 8.7% at 17 nmol/L |
| Ding et al. [89] | Serum 200 μl, PP: MeCN, extraction: SPE Oasis HLB® MeCN; EtOAc Derivatisation (PTAD)/MeCN HPLC: ACQUITY BEH C18® 0.1% CHO2H/H2O/MeNH2; CHO2H/MeOH gradient TMS: ESI+ MRM | Commercial IS 2H6-25OHD3: 613>298, 2H6-25OHD2 625>298, 25OHD3: 607>298, 25OHD2: 619>298 | 2H6-25OHD3: 84.9±2.4%b, 2H6-25OHD2: 79.3±14.4%b | #25OHD3: 0.025, #25OHD2: 0.025 | #25OHD3: 3.8% at 0.025 nmol/L, #25OHD2: 1.6% at 0.025 nmol/L |
| Van den Ouweland et al. [90] | Serum 250 μl, PP: NaOH-MeCN/MeOH (9/1 v/v) SPE: Strata C18-E® H2O-MeOH/H2O (60/40 v/v)-MeOH HPLC: ACQUITY UPLC BEH C18® 0.1% CHO2H/2 mM NH4Ac; MeOH/CHO2H (99.7:0.3 v/v) gradient, TMS: AP-ESI+ SRM | Commercial IS 2H6-25OHD3: 407.5>159.2, 25OHD3: 401.5>159.2, 25OHD2: 413.4>83.1 | 25OHD3: 94.9–106.9% at 49.9–99.9 nmol/L, 25OHD2: 82.7–100.3% at 54.3–108.6 nmol/L | 25OHD3: 3.5, 25OHD2: 2.0, 25OHD3: 1.5, 25OHD2: 1.2 | 25OHD3: 2.7% at 64.9 nmol/L, 25OHD2: 4.2% at 33.3 nmol/L, 25OHD3: 6.0% at 64.9 nmol/L, 25OHD2: 3.8% at 33.3 nmol/L |
| Tai et al. [91] | Serum 2 g pH adjusted to 9.8 (Na2CO3) LLE, extraction: n-hexane/EtAc (50/50 v/v), Residue dissolved in MeOH HPLC: Zorbax CB-CN column H2O/MeOH (34/66 v/v) TMS: APCI+ MRM | Commercial IS 2H3-25OHD3 404>386 2H3-25OHD2 416>398, 25OHD3 C3-epi-25OHD3: 401>383, 25OHD2 C3-epi-25OHD2: 413>395, Stds traceable to NIST | 25OHD3: 100.0–10%, 25OHD2: 98.0–100.1% | 25OHD3: 0.15 ng/g, 25OHD2: 0.1 ng/g | 25OHD3: 0.4% at 6.31 ng/g, 25OHD2: 0.9% 0.86 ng/g, 25OHD3: 0.6% at 6.31 ng/g, 25OHD2: 0.86% 0.86 ng/g |
| Stepman et al. [92] | Serum 250 μl, extraction: LLE: NaOH/n-hexane Sephadex LH-20 chromatography MeOH/CHCl3/cC6H14 (1/4/8, v/v/v) 2-dimensional UPLC Chromatography 1:Acquity BEH 300C4®, column 2: Acquity BEH C18® column-25OHD2 2: Zorbax SB-CN® column-25OHD3, step gradients MeOH/H2O/CHO2H (50/50/0.025) MeOH/H2O/CHO2H (95/5/0.025) TMS: ESI+ SIM | Commercial IS 2H6-25OHD3 407.3>159.3, 2H6-25OHD2 419.4>159.4, 25OHD3: 401.3>159.3, 25OHD2: 413.4>159.4C3-epi-25OHD3 401.3>159.3, Stds Traceable to NIST | 25OHD3: 71%±4%c, 25OHD2: 70%±8%c | 25OHD3: 1.12±0.05, 25OHD2: 1.22±0.05 | 25OHD3: 1.4% at 30.8 nmol/L, 25OHD2: 2.0% at 64.1 nmol/L, 25OHD3: 1.7% at 30.8 nmol/L, 25OHD2: 1.1% at 64.1 nmol/L |
| Adamec et al. [93] | Serum 100 μl, extraction: LLE: Acetone HPLC: ACE3C8® column, gradient: H2O/MeOH+1% toluene TMS: APPI+ MRM | Commercial IS 2H6-25OHD3 407.3>263.3, 2H6-25OHD2 419.3>401.2, 25OHD3: 401.2>257.2, 25OHD2: 413.3>337.2, Stds traceable to NIST | 25OHD3: NR, 25OHD2: NR | 25OHD3: 2.0, 25OHD2: 2.0 | 25OHD3: 3.7% at 5 nmol/L, 25OHD2: 16.7% at 5.0 nmol/L, 25OHD3: 15.4% at 5.0 nmol/L, 25OHD2: 14.0% at 5.0 nmol/L |
| Wang et al. [94] | Plasma 1 ml, PP: MeCN LLE: EtOAc, derivatisation: PTAD HPLC: Hypersil Gold® column MeCN/H2O+0.1% CHO2H gradient (40/60; 60/40; 90/10, 40/60 v/v) TMS: ESI+ MRM | Commercial IS 2H6-25OHD3 564>298, 25OHD3: 558>298 | 25OHD3: 73%±2% (BSA matrix) | 25OHD3: 0.125, 25OHD3: 0.01 | 25OHD3: 2.1% at 25 nmol/L, 25OHD3: 7.0% at 25.0 nmol/L |
| Bogusz et al. [95] | Serum 100 μl, PP: MeOH/MeCN/0.05 M ZnSO4 (6.5/1/2 v/v/v) HPLC: Kinetex C18 NH4CHO2H/MeOH Gradient (70/30; 90/10; 70/30) TMS: APCI+ MRM | Commercial IS 2H6-25OHD3 389>371 389>211, 2H6-25OHD2 401> 383 401>209, 25OHD3: 383>365 383>211, 25OHD2: 395>209 395>269, Stds traceable to NIST | 25OHD3: 98%, 25OHD2: 97% | 25OHD3: 3.0, 25OHD2: 1.5, 25OHD3: 1.5, 25OHD2: 0.5 | 25OHD3: 3% at 41.7 nmol/L, 25OHD2: 4% at 42.1 nmol/L |
| Baecher et al. [96] | Serum 200 μl, PP: MeCN On-line SPE: LiChrospher® column MeOH/H2O (5/95 v/v) HPLC: Kinetex® PFP column MeOH/0.5 mM NH4Ac (75/25 v/v) TMS: APCI+ MRM | Commercial IS 2H6-25OHD3 407.3>263.2, 407.3>159.2, 25OHD3: 401.3>257.2, 401.3>159.2, 25OHD2: 413.4>159.2C3-epi25OHD3 401.3>257.2, 401.3>159.2, NIST SRM 2972 (levels 1-4) used for comparison | 25OHD3: NR, 25OHD2: NR, C3-epi25OHD3, 95.5% at 5.05 nmol/L | 25OHD3: 4.0, 25OHD2: 3.9C3-epi25,OHD3: 2.0 | 25OHD3: 3.1% at 39.8 nmol/L, 25OHD2: 4.9% at 27.5 nmol/L, C3-epi25O,HD3: 4.2% at 20.1 nmol/L 25OHD3: 3.8% at 39.8 nmol/L, 25OHD2: 3.4% at 27.5 nmol/L, C3-epi25O, HD3: 3.4% at 20.1 nmol/L |
| Farrell et al. [97] | Serum 150 μl, PP: 2.0 M ZnSO4/MeOH TMS: 0.2 M/MeOH, SPE:Oasis μElution HLB plate MeOH/H2O (60/40 v/v) 2 mM NH4Ac+0.1% CHO2H/MeOH/2 mM NH4Ac+0.1% CHO2H (27/73 v/v) UPLC: ACQUITY BEH C8® 2 mM NH4Ac+0.1% CHO2H/MeOH/2 mM NH4Ac+0.1% CHO2H Gradient from (27/73 v/v) to (98/2 v/v) ESI+ MRM | Commercial IS 2H6-25OHD3 407.3>159.1, 2H3-25OHD2 416.3>398.3, 25OHD3: 401.3>383.5, 401.3>159.1, 25OHD2: 413.3>83.1, 413.3>395.3 | 25OHD3: NR, 25OHD2: NR | 25OHD3: 2.0, 25OHD2: 2.0, 25OHD3: 0.5, 25OHD2: 0.5 | 25OHD: 1.6% at 79 nmol/L, 25OHD: 2.0% at 79 nmol/L |
| Lensmeyer et al. [98] | Serum 300 μl, PP: MeCN/2 mM ZnSO4 (87/13 v/v)/MeOH, extraction: SPE Strata C18E® MeCN/H2O (45/55 v/v), Acetone/MeCN (20/80 v/v) HPLC: Zorbax cyanopropyl column MeOH/H2O (67/33 v/v), TMS: APCI+ MRM | IS: NR 25OHD3: 383.3>211.1, 25OHD2: 395.3>209.1C3-epi, 25OHD3 383.3>211.1 | NR | NR | NR |
| Thibault et al. [99] | Serum 200 μl, PP: MeCN On-line SPE: X-Terra C18 MeOH/0.1% CHO2H+2 mM NH4Ac in H2O (98/2 v/v)/0.1% CHO2H+2 mM NH4Ac in H2O (68/32 v/v) HPLC: Sunfire C18 MeOH/0.1% CHO2H+2 mM NH4Ac in H2O (98/2 v/v)/ 0.1% CHO2H+2 mM NH4Ac in H2O (85/15 v/v), TMS: ESI+ MRM | Commercial IS [2H6]25-OHD3 407.5>371.3, [2H6]25-OHD2 419.4>355.2, 25OHD3: 401.4>365.3, 25OHD2: 413.4>355.3 | 25OHD3: 4, 25OHD2: 3 | 25OHD3: 3.4% at 59.8 nmol/L, 25OHD2: 1.8% at 99.5 nmol/L, 25OHD3: 5.9% at 66.7 nmol/L, 25OHD2: 5.9% at 101.3 nmol/L | |
| Strathmann et al. [100] | Serum 200 μl extraction: 1 M NaOH/n-heptane HPLC: XTerra MS C8+Restek columns, NH4Ac/0.1% CHO2H in MeOH/H20 (95/5 v/v), TMS: APCI+ MRM | Commercial IS [2H6]25-OHD3 407.3>371.3, [2H6]25-OHD2 419.4>355.2, 25OHD3: 401.3>355.3, 25OHD2: 413.4>355.3, Stds traceable to NIST | 25OHD3: 80–116% (23.4 nmol/L), 25OHD2: 94–115% (23.4 nmol/L) | 25OHD3: 1.95, 25OHD2: 0.6 | 25OHD3: 2.9% at 58 nmol/L, 25OHD2: 2.8% at 85 nmol/L, 25OHD3: 9.6% at 63 nmol/L, 25OHD2: 6.2% at 95 nmol/L |
| Mochizuki et al. [101] | Serum or plasma 25 μl, PP: MeCN 2-dimension HPLC: SPE: Turboflow XL C18-P® column, step gradient 0.1% CHO2H; MeCN/propanol-2ol/acetone (44/40/20 v/v/v); MeOH/0.1% CHO2H HPLC: Hypersil Gold® column 0.1% CHO2H; MeOH/0.1% CHO2H TMS: APCI+ SRM | Commercial IS [2H6]25-OHD3 389.3>263.2 25OHD3: 383.3>365.2 25OHD2: 395.3>377.4 Stds traceable to NIST | 25OHD3: 102.6–106% (36.9–59.8 nmol/L), 25OHD2: NR | 25OHD3: 2.2, 25OHD2: 3.5, 25OHD3: 0.8, 25OHD2:2.2 | 25OHD3: 5.2% at 18 nmol/L, 25OHD2: 10.6% at 18 nmol/L, 25OHD3: 7.2% at 18 nmol/L, 25OHD2: NR |
| Zhang et al. [102] | Serum 200 μl, PP: MeOH, extraction: n-heptane HPLC: Zorbax SB-C18, Step Gradient: 2 mM NH4Ac/0.1% CHO2H–H2O; 2 mM NH4Ac/0.1% CHO2H–MeOH, TMS: ESI+ MRM | Commercial IS [2H3]-25OHD3 404.3>368.2, [2H3]-25OHD2 416.3>358.2, 25OHD3: 401.3>365.2, 25OHD2: 413.3>355.2 | 25OHD3: ≥62% (125–200 nmol/L), 25OHD2: ≥72% (18–200 nmol/L) | 25OHD3: 6.2, 25OHD2: 6.2, 25OHD3: NR, 25OHD2: NR | 25OHD3: 2.2% at 18 nmol/L, 25OHD2: 2.1% at 18 nmol/L, 25OHD3: 4.4% at 18 nmol/L, 25OHD2: 5.0 at 18 nmol/L |
| Kaufmann et al. [103] | Serum 100 μl, PP: 0.1 M HCl/0.2 M ZnSO4/MeOH, extraction: n-hexane/t-butyl ether (1/1 v/v), derivatisation (DMEQ-TAD)/AcOEt UPLC: BEH-Phenyl column, MeOH/H2O gradient, TMS: ESI+ MRM | Commercial IS 2H3-25OHD3: 613>298, 2H3-25OHD2 625>298 25OHD3: 746.6>468, 25OHD2: 758.6>468 | 25OHD3: NR, 25OHD2: NR | 25OHD3: 0.25, 25OHD2: 0.25, 25OHD3: 0.10, 25OHD2: 0.10 | 25OHD3: 3–4% at 55 nmol/L, 25OHD2: 3–4% at 83 nmol/L, 25OHD3: 4–7% at 55 nmol/L, 25OHD2: 4–7% at 83 nmol/L |
HPLC: high performance liquid chromatography; UPLC: UlLC: performance liquid chromatography; MS: mass spectrometry; TMS: tandem-mass spectrometry; AP: atmospheric pressure; ESI: electron spray ionization; APCI: atmospheric pressure chemical ionization; APPI: atmospheric pressure photo-ionization; ID: isotope dilution; MRM: multiple reaction monitoring; SRM: selected reaction monitoring; PP: protein precipitation; SPE: solid phase extraction; LLE: liquid/liquid extraction; OLTFE: on-line turboflow extraction; DMEQ-TAD: 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione; NPTAD: 4-(4-Nitrophenyl)-1,2,4-triazoline-3,5-dione; PTAD: 4-phenyl-1,2,4-triazoline-3,5-dione; EAD: enzyme-assisted derivatisation; GP: Girard reagent P reagent (1-(carboxymethyl)pyridinium chloride hydrazide); 25OHdC: 25-Hydroxy-7-dehydrocholesterol; 1α–OHD3. 1-alpha-hydroxyvitamin D3 AcOEt: ethyl acetate; MeNH2: methyl amine; MeOH: methanol; EtOH: ethanol; NH4Ac: ammonium acetate; MeCN: acetonitrile: Et2O: diethyl-ether; KOH: potassium hydroxide; MeCl2: dichloromethane; HClO4: perchloric acid; THF: tetrahydrofuran; ASC: 20% ascorbic acid water solution; CHO2H: formic acid; IS: internal standard; NR: not reported; NIST: National Institute of Standards and Technology (Gaithersburg, USA); SRM: standard reference material; levels 1–4: level 1: human serum; level 2: human serum diluted with horse serum to achieve a lower 25(OH)Dx concentration; level 3: human serum fortified with 25(OH)D2; and level 4: human serum fortified with 3-epi-25(OH)D3.
a
Recovery: exogenously added vitamin D metabolite.
b
% Recovery±SD for the 2 deuterated compounds at a 50 fmol/μl fortification level.
c
Expressed as % recovery of the NIST-certified values.