Quantitation of Four Chiral Drug Compounds Simultaneously Using NormalPhase LC-MS/MS Equipped With an APPI Ion SourceAuthors: Min Huang, Larry M. Mallis, and Zhongping (John) Lin

IntroductionChiral separation is important but also challenging in bioanalytical plasma analysis. The investigation of luliconazole efficacy requires the measurement of its two geometric isomers (E/Z or trans/cis forms), as well as their chiral (R and S) forms. To measure the individual concentrations of the four chiral compounds by LC-MS/MS, it is necessary to chromatographically separate all four enantiomers.. As expected, baseline separation was only achieved between E and Z forms with non-chiral columns. With chiral columns, good separation was achieved between RE and SE, or RZ and SZ, separately, but not all of the chiral compounds.

MethodIn this work, APPI ion source was used to replace the typical ion sources of ESI or APCI. The advantage was that APPI is well suited to the organic solvents (e.g., hexane), which eliminates the requirement of post column aqueous addition necessary for ESI or APCI. Furthermore, the organic solvents themselves are good dopants for APPI and lead to high sensitivity of LC-APPI-MS/MS.

Sample preparation procedure was developed which uses 100 µL plasma and 0.5 ng lanoconazole as internal standard. LLE is performed using 1.0 mL MTBE. The extract is blown down to dryness at 37 °C and reconstituted in 100 uL ethanol for injection.

ResultsIn this work, normal phase LC was investigated. After many normal phase columns were investigated, an Lux column was found to be hopeful although with a 35 min runtime. Further optimization led to achieving baseline separation of all four enantiomers with a 15 min runtime. Normal phase solvents are not a good match with ESI or APCI. It has been previously reported that water was added post column to allow applying the high voltage that is required for ESI. In practice, it was very difficult to make the aqueous addition homogenous, thus, as a result signal fluctuation was observed which significantly deteriorated the quantitative measurement.

References1. Mathieu Gaudin, Laurent Imbert, Danielle Libong, Pierre Chaminade, Alain Brunelle, David Touboul, and Olivier Laprevote, “Atmospheric Pressure Photoionization as a Powerful Tool for Large-Scale LipidomicStudies”, J. Am. Saoc. Mass Spectrom, 2012, 23, 869-879.2. Brenna Black, Chenxing Sun, Yuan Yuan Zhao, Michael Ganzle, and Jonathan Curtis, “Antirfungal Lipids Produced by Lactobacilli and their Structural Identification by Normal Phase LC/Atmospheric Pressure Photoionization-MS/MS”, J. Agric. Food Chem. 2013, 61, 5338-5346.3. Christian Grun and Sophie Besseau, “Normal-phase liquid chromatography-atmospheric-pressure photoionization-mass spectrometry analysis of cholesterol and phytosterol oxidation products”, J. Chrom. A, 2015, in press.4. Ralf Dieter Josephs, Adeline Daireaux, Ttiphaine Choteau, Steven Westwood, and Roberty Ian Wielgosz, “Normal phase-liquid chromatography-tandem mass spectrometry with atmospheric pressure photoionization for the purity assessment of 17β-estradiol”, Anal. Bioanal. Chem., 2015, 407, 3147-3157.

Preliminary Column EvaluationSciex Photospray Source

Normal Phase HPLC & MS/MS ConditionsHPLC Conditions Description

HPLC Shimadzu LC20AD

Mobile Phase A Ethanol

Mobile Phase B Hexane

Autosampler Temp Ambient

Separation Type Gradient

Column Phenomenex Lux,4.6 x 100 mm, 3.0 µm

Flow Rate 0.90 mL/min

Injection Volume 20 µL

Sciex API 5000 Photospray

Ionization Mode Positive

Assay Range 50 – 10,000 pg/mL

Sample Treatment Liquid Liquid Extraction

Anticoagulant K2EDTA

Time (Minutes)

Flow Rate (mL/min)

%B

0 0.9 80

0.5 0.9 80

1.0 0.9 70

9.3 0.9 66

9.31 1.5 60

12.7 1.5 60

12.71 1.5 80

14.2 1.5 80

14.21 0.9 80

15.0 Stop

MS Conditions Description

Mass Spectrometer Sciex API 5000

Ion Source Photospray

CUR 10

GS1 38

GS2 25

IS 780

TEM 280 oC

DP 150

EP 10

CE 35

CXP 20

Luliconozole Enantiomers 354 150

Lanaconozole IS Isomers 322 150

Sample Extraction Procedure• Pipet 100 µL of plasma into a 16 x 100 mm glass tube.

• Add 600 µL of pre-chilled 1% formic acid in H2O solution (4°C) into all above samples, Vortex for approximately 30 seconds.

• Immediately add 3 mL of Extraction Solution (60:40 MTBE: Ethyl Acetate) into all above sample tubes, cap and vortex vigorously for 10 minutes..

• Centrifuge all above sample tubes for 5 minutes at 3000 rpm, ambient temperature.

• Flash freeze the aqueous layer at the tube bottom in an acetone-dry ice bath. Transfer extracted supernatant into a 12x75 mm glass tube.

• Add 10 µL of DMSO to each 12x75 mm glass tube. Dry down the samples under N2 at 30oC water bath (~ 25 minutes) at the speed (≤ 10psi).

• To each tube, added 100 µL of Reconstitution Solution. Cap and vortex for 1 minute.

• Centrifuge all above sample tubes for 1 minute at 3000 rpm, ambient temperature.

• Transfer the clear supernatant into a HPLC vial. Inject 20 µL.

N

N

N

S

S

Cl

S

S

N

N

N

Cl

Internal Standard Structures

Example Chromatogram and Standard Curve for RZ

Example Chromatogram and Standard Curve for SE

Example Chromatogram and Standard Curve for RE

Example Chromatogram and Standard Curve for SZ

Watson Run IDIntra-Run QC-LLOQ 50

pg/mLIntra-Run QC-Low

0.15 ng/mLIntra-Run QC-Mid

3.00 ng/mLIntra-Run QC-High

7.50 ng/mL

1

0.0570 0.162 3.092 7.447

0.0540 0.153 3.037 7.461

0.0540 0.154 3.051 7.431

0.0560 0.152 3.051 7.346

0.0540 0.158 3.03 7.381

0.0550 0.151 3.036 7.295

Intra-run Mean 0.055 0.155 3.05 7.39

Intra-run SD 0.00126 0.00420 0.0225 0.0647

Intra-run %CV 2.3% 2.7% 0.7% 0.9%

Intra-run %Bias 10.0% 3.3% 1.7% -2.1%

n 6 6 6 6

2

0.0562 0.151 2.96 7.37

0.0553 0.148 2.95 7.35

0.055 0.149 2.91 7.28

0.0532 0.151 2.94 7.34

0.0516 0.141 2.96 7.27

0.0534 0.146 2.92 7.28

Intra-run Mean 0.0541 0.148 2.94 7.32

Intra-run SD 0.00169 0.00378 0.0210 0.0432

Intra-run %CV 3.1% 2.6% 0.7% 0.6%

Intra-run %Bias 8.2% -1.6% -2.0% -3.1%

n 6 6 6 6

3

0.051 0.146 2.97 7.27

0.0529 0.149 2.98 7.39

0.0522 0.146 2.99 7.41

0.0514 0.149 3.02 7.64

0.051 0.15 2.98 7.34

0.0518 0.149 2.98 7.47

Intra-run Mean 0.0517 0.148 2.99 7.42

Intra-run SD 0.000744 0.00172 0.0175 0.127

Intra-run %CV 1.4% 1.2% 0.6% 1.7%

Intra-run %Bias 3.4% -1.2% -0.4% -1.7%

n 6 6 6 6

Inter-run Mean 0.0536 0.1503 2.9921 7.3762

Inter-run SD 0.00187 0.00470 0.05000 0.09295

Inter-run %CV 3.5% 3.1% 1.7% 1.3%

Inter-run %Bias 7.2% 0.2% -0.3% -1.7%

n 18 18 18 18

Watson Run IDIntra-Run QC-LLOQ

50 pg/mLIntra-Run QC-Low

0.15 ng/mLIntra-Run QC-Mid

3.00 ng/mLIntra-Run QC-High

7.50 ng/mL

1

0.0559 0.155 2.99 7.35

0.0537 0.154 3.02 7.37

0.0552 0.157 2.99 7.38

0.0561 0.149 2.97 7.42

0.0537 0.152 3.02 7.38

0.0536 0.147 3.02 7.29

Intra-run Mean 0.0547 0.152 3.00 7.37

Intra-run SD 0.00117 0.00378 0.0214 0.0432

Intra-run %CV 2.1% 2.5% 0.7% 0.6%

Intra-run %Bias 9.4% 1.6% 0.1% -2.5%

n 6 6 6 6

2

0.0529 0.15 2.96 7.42

0.0519 0.146 2.97 7.41

0.0511 0.151 2.94 7.38

0.0552 0.149 2.98 7.42

0.0508 0.148 2.96 7.33

0.0521 0.145 2.93 7.39

Intra-run Mean 0.0523 0.148 2.96 7.39

Intra-run SD 0.00159 0.00232 0.0186 0.0343

Intra-run %CV 3.0% 1.6% 0.6% 0.5%

Intra-run %Bias 4.7% -1.2% -1.4% -2.1%

n 6 6 6 6

3

0.0461 0.143 2.98 7.31

0.0476 0.145 2.98 7.37

0.0458 0.143 3.01 7.4

0.0479 0.147 3.04 7.66

0.0473 0.145 2.98 7.36

0.0463 0.142 2.96 7.47

Intra-run Mean 0.0468 0.144 2.99 7.43

Intra-run SD 0.000876 0.00183 0.0286 0.125

Intra-run %CV 1.9% 1.3% 1.0% 1.7%

Intra-run %Bias -6.3% -3.9% -0.3% -1.6%

n 6 6 6 6

Inter-run Mean 0.0513 0.148 2.98 7.40

Inter-run SD 0.00359 0.00431 0.0295 0.0788

Inter-run %CV 7.0% 2.9% 1.0% 1.1%

Inter-run %Bias 2.6% -1.2% -0.6% -1.4%

n 18 18 18 18

Watson Run ID Intra-Run QC-LLOQ 50 pg/mL

Intra-Run QC-Low 0.15 ng/mL

Intra-Run QC-Mid 3.00 ng/mL

Intra-Run QC-High 7.50 ng/mL

1

0.0527 0.156 3.07 7.49

0.053 0.15 3.08 7.55

0.055 0.153 3.03 7.49

0.0571 0.15 3.02 7.49

0.0534 0.151 3.09 7.5

0.0505 0.149 3.08 7.38

Intra-run Mean 0.0536 0.152 3.06 7.48

Intra-run SD 0.00224 0.00259 0.0293 0.0557

Intra-run %CV 4.2% 1.7% 1.0% 0.7%

Intra-run %Bias 7.2% 1.0% 2.1% -0.9%

n 6 6 6 6

2

0.0527 0.146 2.98 7.38

0.0514 0.141 2.98 7.33

0.0517 0.148 2.93 7.28

0.051 0.149 2.99 7.36

0.0488 0.144 2.98 7.27

0.0493 0.142 2.91 7.35

Intra-run Mean 0.0508 0.145 2.96 7.33

Intra-run SD 0.00149 0.00322 0.0331 0.0445

Intra-run %CV 2.9% 2.2% 1.1% 0.6%

Intra-run %Bias 1.6% -3.3% -1.3% -2.9%

n 6 6 6 6

3

0.0469 0.142 3.01 7.3

0.0477 0.145 2.98 7.3

0.0467 0.142 3.01 7.35

0.0483 0.146 3.01 7.6

0.0478 0.144 2.96 7.31

0.0477 0.14 2.96 7.36

Intra-run Mean 0.0475 0.1432 2.9883 7.3700

Intra-run SD 0.00060 0.00223 0.02483 0.11559

Intra-run %CV 1.3% 1.6% 0.8% 1.6%

Intra-run %Bias -5.0% -4.6% -0.4% -2.4%

n 6 6 6 6

Inter-run Mean 0.0507 0.147 3.00 7.39

Inter-run SD 0.00297 0.00448 0.0515 0.0998

Inter-run %CV 5.9% 3.1% 1.7% 1.4%

Inter-run %Bias 1.3% -2.3% 0.1% -1.4%

n 18 18 18 18

Watson Run IDIntra-Run QC-LLOQ

50 pg/mLIntra-Run QC-Low

0.15 ng/mLIntra-Run QC-Mid

3.00 ng/mLIntra-Run QC-High

7.50 ng/mL

1

0.0507 0.149 3.02 7.31

0.0509 0.150 3.01 7.46

0.0501 0.153 3.09 7.54

0.0493 0.150 3.09 7.56

0.0499 0.148 3.05 7.53

0.0488 0.144 3.07 7.47

Intra-run Mean 0.0500 0.149 3.06 7.48

Intra-run SD 0.000804 0.00297 0.0345 0.0915

Intra-run %CV 1.6% 2.0% 1.1% 1.2%

Intra-run %Bias -0.1% -0.7% 1.8% -0.9%

n 6 6 6 6

2

0.0522 0.147 3.02 7.39

0.0500 0.146 3.02 7.24

0.0508 0.147 3.03 7.12

0.0489 0.150 2.96 7.35

0.0489 0.149 2.94 7.37

0.0490 0.147 3.01 7.44

Intra-run Mean 0.0500 0.148 3.00 7.32

Intra-run SD 0.00133 0.00151 0.0372 0.118

Intra-run %CV 2.7% 1.0% 1.2% 1.6%

Intra-run %Bias -0.1% -1.6% -0.1% -3.1%

n 6 6 6 6

3

0.0476 0.145 2.95 7.34

0.0494 0.147 2.99 7.45

0.0516 0.145 2.97 7.24

0.0525 0.140 2.97 7.45

0.0517 0.149 2.97 7.32

0.0477 0.146 2.95 7.33

Intra-run Mean 0.0501 0.145 2.97 7.36

Intra-run SD 0.00215 0.00301 0.0151 0.0817

Intra-run %CV 4.3% 2.1% 0.5% 1.1%

Intra-run %Bias 0.2% -3.1% -1.1% -2.6%

n 6 6 6 6

Inter-run Mean 0.0500 0.147 3.01 7.38

Inter-run SD 0.00144 0.00289 0.0474 0.116

Inter-run %CV 2.9% 2.0% 1.6% 1.6%

Inter-run %Bias 0.0% -1.8% 0.2% -1.5%

n 18 18 18 18

Precision and Accuracy Data (Intra & Inter) for RZ

Precision and Accuracy Data (Intra & Inter) for SE

Precision and Accuracy Data (Intra & Inter) for RE

Precision and Accuracy Data (Intra & Inter) for SZ

N

N

N

S

S

Cl

Cl

S

S

Cl

N

N

N

Cl

N

N

N

S

S

Cl

Cl

S

S

Cl

N

N

N

Cl

Isomer Structures

Example Blank and Double Blank Chromatograms

Isomer StandardNominal (ng/mL)

Day 1 Day 2 Day 3 Mean SD %CV %Bias

RZ

std 1 0.0500 0.049 0.0508 0.0507 0.0502 0.00101 2.0% 0.3%

std 2 0.100 0.104 0.0974 0.0974 0.100 0.00381 3.8% -0.4%

std 3 0.400 0.379 0.402 0.397 0.393 0.0121 3.1% -1.8%

std 4 1.00 0.965 0.946 0.986 0.966 0.0200 2.1% -3.4%

std 5 2.00 1.942 1.93 2.04 1.97 0.0603 3.1% -1.5%

std 6 5.00 5.008 5.03 5.05 5.03 0.0210 0.4% 0.6%

std 7 8.00 8.11 8.32 8.06 8.16 0.138 1.7% 2.0%

std 8 10.0 10.738 10.5 9.98 10.4 0.388 3.7% 4.1%

SZ

std 1 0.0500 0.0505 0.0506 0.0507 0.0506 0.000100 0.2% 1.2%

std 2 0.100 0.0992 0.0985 0.0976 0.0984 0.000802 0.8% -1.6%

std 3 0.400 0.385 0.39 0.392 0.389 0.00361 0.9% -2.8%

std 4 1.00 0.963 0.966 1.01 0.980 0.0263 2.7% -2.0%

std 5 2.00 1.96 1.96 2.03 1.98 0.0404 2.0% -0.8%

std 6 5.00 5.04 4.99 4.95 4.99 0.0451 0.9% -0.1%

std 7 8.00 8.08 8.42 8.10 8.20 0.191 2.3% 2.5%

std 8 10.0 10.8 10.3 10.1 10.4 0.361 3.5% 4.0%

RE

std 1 0.0500 0.0494 0.0507 0.051 0.0504 0.000850 1.7% 0.7%

std 2 0.100 0.104 0.0975 0.096 0.0992 0.00425 4.3% -0.8%

std 3 0.400 0.379 0.399 0.395 0.391 0.0106 2.7% -2.3%

std 4 1.00 0.98 0.972 1.01 0.987 0.0200 2.0% -1.3%

std 5 2.00 1.98 1.95 2.02 1.98 0.0351 1.8% -0.8%

std 6 5.00 4.94 4.99 4.99 4.97 0.0289 0.6% -0.5%

std 7 8.00 8.19 8.34 8.04 8.19 0.150 1.8% 2.4%

std 8 10.0 10.5 10.3 10.1 10.3 0.200 1.9% 3.0%

SE

std 1 0.0500 0.0499 0.0505 0.0496 0.0500 0.000458 0.9% 0.0%

std 2 0.100 0.101 0.0986 0.102 0.101 0.00175 1.7% 0.5%

std 3 0.400 0.39 0.391 0.396 0.392 0.00321 0.8% -1.9%

std 4 1.00 0.99 0.998 0.985 0.991 0.00656 0.7% -0.9%

std 5 2.00 1.97 1.98 2.02 1.99 0.0265 1.3% -0.5%

std 6 5.00 5 5 5.08 5.03 0.0462 0.9% 0.5%

std 7 8.00 8.14 8.02 7.84 8.00 0.151 1.9% 0.0%

std 8 10.0 10.2 10.3 10.1 10.2 0.100 1.0% 2.0%

Precision and Accuracy Inter-day Data for Standard Curves

ConclusionsUnder the optimized conditions, the chiral compounds were baseline separated and eluted at 5.4, 7.5, 8.3 and 11.4 min, respectively. The internal standard was also separated as two chiral forms, with RT at 9.0 and 11.1 min. It was demonstrated that better accuracy was obtained by applying the second IS for correcting the last chiral compound, while using the IS at 9.0 min for the first three chiral compounds. The LLOQ was 50 pg/mL for all four chiral compounds.