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LC ESI/MS reference measurement procedure for HbA 2 Donatella Caruso Dept. Pharmacological and Biomolecular Sciences Università degli Studi Milano
LC ESI/MS reference measurement procedure for HbA2
Donatella Caruso Dept. Pharmacological and Biomolecular Sciences Università degli Studi Milano
The determination of HbA2 is accepted as the gold standard in the screening for β-thalassemia carriers.
Highly accurate and precise measurements are needed, also because the difference between normal and pathological HbA2 values is slight.
Fetal hemoglobin: assessment of glyca3on and acetyla3on status by electrospray ioniza3on mass spectrometry Andrew S. Davison, Brian N. Green and Norman B. Roberts Clin Chem Lab Med 2008;46(9):1230–1238
….Here, we describe the evaluation of ESI-MS for measurement of glycated (GHbF) and acetylated (AcHbF) fetal hemoglobin and the identification by mass of different chains of fetal hemoglobin………..
AIM: To develop a reference measurement procedure based on the quantification of intact globin chains by LC-ESI/MS
I STEP: CHOICE OF THE ANALYTICAL CONDITIONS
II STEP: VALIDATION OF THE METHOD II STEP: VALIDATION OF THE METHOD
III STEP: INTERLABORATORY COMPARISON III STEP: INTERLABORATORY COMPARISON
Sample prepara>on
Desal>ng on ca>on exchange resin bead
Removal of WBC, platelets, plasma Filtra>on through cellulose column
Lysis with H2O/CCl4
Removal of cellular stroma
Dilu>on with acetonitrile/ formic acid
centrifuga>on
Samples for MS Hb 1 mg/mL Acetonitrile 50 % Formic acid 0.18 %
Davison AS et al. Clin Chem Lab med
2008;46:1230 (modified)
Fresh blood in EDTA
RBC
Hemolysate
Instrumentation: ESI-linear ion trap (LTQ, Thermofisher, USA)
Vydac C4 250x4,6mm Column:
Surveyor LC Pump Method -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐ Eluents
A: H20-‐CH3CN 4:1+0.05% TFA B: H20-‐CH3CN 2:3+0.05% TFA Program Time (min) Flow (mL/min) A (%) B (%) -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐ -‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐ -‐-‐-‐-‐-‐ -‐-‐-‐-‐-‐ 0.00 0.800 48.0 52.0 2.00 0.800 48.0 52.0 13.00 0.800 42.0 58.0 16.00 0.800 42.0 58.0 25.00 0.800 31.0 69.0 30.00 0.800 48.0 52.0 35.00 0.800 48.0 52.0
Analytical conditions
RT: 11.00 - 27.00 SM: 7G
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27Time (min)
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
RT: 23.55MA: 30189496
RT: 15.81MA: 18684699
RT: 17.76MA: 676534
23.48
15.71
24.20
24.42
24.6224.87
25.32
17.49 22.5519.3818.06 19.51 22.2615.0712.71 21.0713.81
12.19
NL:1.04E6TIC MS 2THcolbis
ß/α=0.62
δ/α=0.022
TIC trace of a β-thalassemic subject hemoglobin
Only few words on ESI-‐ion trap
mass spectrometry….
The general term "atmospheric pressure ionization" (API) includes the most notable technique, electrospray
ionization (ESI)
The gas phase peptide ions can be generated from the online eluate of a LC column or from direct injection
ESI is able to produce single or multiple charged ions, depending on several factors.
Hb_39a #1804-1953 RT: 18.86-19.79 AV: 150 NL: 4.16E5F: ITMS + c ESI sid=10.00 Full ms [600.00-1500.00]
600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
1164.781081.68
1009.66
1261.74
946.65
891.03
1376.32841.63
797.40
757.60
721.60 954.31 1274.931177.30898.16 1400.10688.84 963.01636.51 1316.691215.16 1446.73
α chain
Since MS measure the mass-to-charge (m/z) ratio, the ESI mass spectrum for a large molecule typically contains multiple signals corresponding to the different charge states.
S#: 1 RT: 0.01 AV: 1 NL: 1.54E7T: + p Full ms
700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
Z=24
Z=15
Z=14
Z=13
Z=12
Z=11
Z=10
Z=9
1060.5
1131.1 1211.9998.2
942.91305.0
893.31413.5848.6
808.21541.9
771.5
1696.0
616.2
1884.2707.31420.7 1888.91225.4 1315.8 1820.81551.7679.1 1706.1 1938.7
S#: 1 RT: 0.01 AV: 1 NL: 1.23E8T: + p Full ms
16800 16850 16900 16950 17000 17050 17100 17150mass
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
16951.0
16995.0
16933.0 17011.0 17031.017090.016784.0 17114.0 17142.016894.016802.0 16851.0
The target molecules are ionized into multiple charge states producing a waveform spectrum that can be de-convoluted into parent peaks.
gamma chain m/z
alpha chain m/z
1062 946 1138 1009
2 MULTICHARGED ION DETECTED Hb_39a #1804-1953 RT: 18.86-19.79 AV: 150 NL: 4.16E5F: ITMS + c ESI sid=10.00 Full ms [600.00-1500.00]
600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
1164.781081.68
1009.66
1261.74
946.65
891.03
1376.32841.63
797.40
757.60
721.60 954.31 1274.931177.30898.16 1400.10688.84 963.01636.51 1316.691215.16 1446.73
α chain
Hb_74b #1179-1267 RT: 13.66-14.30 AV: 89 NL: 3.10E4F: ITMS + c ESI sid=10.00 Full ms [600.00-1500.00]
600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
1138.70
1062.85
1226.18
996.54
1328.21
937.99
885.97
1448.81
839.35
697.02636.55
797.48
731.84 1103.39 1152.431030.01 1252.58965.64908.931376.67 1472.46642.87
δ chain
RT: 7.00 - 24.00 SM: 13G
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Time (min)
10 20 30 40 50 60 70 80 90 100
Rel
ativ
e A
bund
ance
10 20 30 40 50 60 70 80 90 100
Rel
ativ
e A
bund
ance
RT: 20.08 MA: 13192096
RT: 15.04 MA:507942
α
δ
Calibrator 2 multicharge-
HbA2 = 3.9 Theoretical value = 4.3
10 MULTICHARGED IONS HAS BEEN DETECTED
10 Multicharge
δ/α = 0.043
RT: 7.00 - 24.00 SM: 13G
8 10 12 14 16 18 20 22 24 Time (min) 10 20 30 40 50 60 70 80 90 100
Rel
ativ
e A
bund
ance
10 20 30 40 50 60 70 80 90 100
Rel
ativ
e A
bund
ance
RT: 20.09 MA:63301231
RT: 14.92 MA: 2752548
10 multicharged- calibrator α
δ
HbA2 = 4.3% Theoretical value = 4,3%
gamma chain m/z
alpha chain m/z
797 757 839 797 885 841 937 890 996 946 1062 1009 1138 1081 1225 1164 1327 1262 1448 1375
II STEP: VALIDATION OF THE METHOD
Samples
Human frozen hemolysates treated to remove most of the salt adducts with proteins (no plasma, no leukocytes, no platelets).
4 calibrators (HbA2 concentration determined by Tosoh G8 WHO calibrated; 2.5, 3.4, 5.6 and 6.2%)
12 samples to be analyzed in triplicate 4 calibrators (HbA2 concentration determined by Tosoh G8 WHO calibrated ; 2.5,
3.6, 4.9 and 6.3%) 18 samples to be analyzed in triplicate
15
Second batch: sample 17-38
First batch: samples 1-16
Third batch: samples 39-78
4 calibrators (HbA2 concentration determined by Tosoh G8 WHO calibrated ; 2.4, 3.3, 5.1 and 6.1%)
35 samples to be analyzed in triplicate
y = 0,0063x + 0,0052 R² = 0,99647
0,000 0,005 0,010 0,015 0,020 0,025 0,030 0,035 0,040 0,045 0,050
0 1 2 3 4 5 6 7
I batch
y = 0,0068x + 0,0014 R² = 0,99892
0,000 0,005 0,010 0,015 0,020 0,025 0,030 0,035 0,040 0,045 0,050
0 1 2 3 4 5 6 7
II batch
y = 0,0061x - 0,0005 R² = 0,99933
0,000
0,005
0,010
0,015
0,020
0,025
0,030
0,035
0,040
0 1 2 3 4 5 6 7
III batch
The calibra>on curve parameters
HbA2 (%)
HbA2 (%)
Batch r2 slope (x103) 1 0,997 6,5 2 0,999 6,3 3 0,998 6,1
mean 0,998 6,300 SD 0,001 0,200
CV% 0,10 3,17
RT: 7.00 - 24.00 SM: 13G
8 10 12 14 16 18 20 22 24 Time (min)
10 20 30 40 50 60 70 80 90
100
Rel
ativ
e A
bund
ance
10 20 30 40 50 60 70 80 90
100 R
elat
ive
Abu
ndan
ce
RT: 20.48 MA: 10252087
22.29 13.51 15.08 19.38 15.76 12.89 17.44 10.34 9.37 7.73
RT: 15.18 MA: 396872
20.55
13.53 21.74 13.01 16.33 12.26 22.55 16.84 19.59 10.22 8.84
NL: 2.14E5 m/z= 756.80-757.80+796.60-797.60+840.80-841.80+ 890.20-891.20+945.80-946.80+1008.80-1009.80+ 1080.80-1081.80+1163.90-1164.90+1261.00-1262.00+ 1375.30-1376.30 F: ITMS + c ESI sid=10.00 Full ms [600.00-1500.00] MS Hb_051010_10b
NL: 2.05E4 m/z= 796.70-797.70+838.60-839.60+885.20-886.20+ 937.10-938.10+995.60-996.60+1062.00-1063.00+ 1137.80-1138.80+1225.20-1226.20+1327.20-1328.20+ 1447.80-1448.80 F: ITMS + c ESI sid=10.00 Full ms [600.00-1500.00] MS Hb_051010_10b
10 Multicharge - sample
δ
α
1st batch
2nd batch
MS precision profile
3rd batch
Rela>onship between HbA2 measured by the MS and HPLC techniques
III STEP: INTERLABORATORY COMPARISON
TAKE-‐HOME MESSAGE
home
The possibility to perform the quan3ta3ve determina3on of HbA2 without protein diges3on, has been explored.
ESI-‐MS of the intact Hb chains appears to be a viable method for determining the δ-‐chain and hence detec3ng β-‐thalassemia trait in blood samples.
acknowledgment Renata Paleari Federico Abbiati Andrea Mosca Flavio Giavarini
all the HbA2 working group
