Peak-purity by LC-MS and LC-DAD

Knut DyrstadErlend Hvattum

Sharon JaraArnvid Lie

Peak-purity

• Peak-purity is of vital importance for documenting the safety and the efficacy of a pharmaceutical drug

• The improvement of the chemical syntheses and the pharmaceutical processes requires knowledge about purity

• LC-MS is the matter of choice (standalone equipments as MS, NMR and IR are frequently used to explore composition)

• Among all samples being analyzed, the challenge is to scientifically evaluate which samples need the extra purity attention

• The supplementary use of LC-MS and LC-DAD-PCA (chemometrics) in purity investigations will be discussed

Peak-purity assesment by LC-MS

• Traditionally it is a manual task; i.e. time consuming

• Different scans accross the peak is evaluated to determine whether co-eluting compounds are present

• Ideally, both negative and positive ESI should be employed

ExampleHPLC-parametersMobile phases: A = 0.1% TFA in water; B = 0.085% TFA in MeCNColumn: Symmetry C18, 3.5, 4.6x150mm Flow: 0.9 ml/minUV: 200 – 400 nmColumn temp.: 30 ºC;

MS-parametersQTof-micro MS (Waters)Positive ion ESI mode Capillary spray at 3kVTemperatures: 100º (source) and 300º (dessolvation)Cone voltage: 30VScan range: m/z 100 – 2000 and 2 secs/per scan

SampleExact mass of compound = 1257.49 uConcentration = 2.12 mg/ml in water

NC100717 (2.12 mg/ml 17feb10); FKAL 420-014; CV30

Time6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

%

16

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00

AU

1.0e-1

2.0e-1

3.0e-1

4.0e-1

ehv qtof 10021704 3: Diode Array Range: 1.323e+115.32

15.11

14.87

9.49 14.23

18.71

16.80

ehv qtof 10021704 1: TOF MS ES+ TIC

4.76e315.36

9.506.814.73 5.79 11.23 13.5618.7118.04

22.6321.8221.01 23.30

NC100717 (2.12 mg/ml 17feb10); FKAL 420-014; CV30

Scan410 415 420 425 430 435 440 445 450 455 460 465 470 475 480

%

15

ehv qtof 10021704 1: TOF MS ES+ TIC

6.49e3

1 2 3 4 5 6

UV-chromatogram

Mass chromatogram

NC100717 (2.12 mg/ml 17feb10); FKAL 420-014; CV30

m/z200 400 600 800 1000 1200 1400 1600 1800

%

0

100

ehv qtof 10021704 435 (15.359) Sm (Mn, 1x3.00); Cm (430:460-161:237) 1: TOF MS ES+ 2.00e3629.8

420.2

158.0

214.1

405.2

420.5

420.9

630.3 1258.6

632.3

632.8

640.8

641.3

1259.6

1261.6

1262.6

[M + 2H]2+

[M + H]+

[M + 3H]3+

NC100717 (2.12 mg/ml 17feb10); FKAL 420-014; CV30

m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

ehv qtof 10021704 460 (16.242) Cm (460:465-408) 1: TOF MS ES+ 56158.0

215.11260.6415.2304.0

630.8537.9 1142.31087.0942.9723.8 1995.51871.01837.31687.51652.6

ehv qtof 10021704 454 (16.031) Cm (454:459-408) 1: TOF MS ES+ 56629.8214.1

215.1628.9216.1 420.2399.3

630.3 1258.6632.6

648.8

1258.61260.5

1261.6

ehv qtof 10021704 448 (15.818) Cm (448:453-408) 1: TOF MS ES+ 56629.8214.1 420.2

405.2215.1

420.2

629.6

630.3 1258.6 1258.6

1262.7

ehv qtof 10021704 442 (15.607) Cm (442:447-408) 1: TOF MS ES+ 56629.8420.2214.1 405.2

627.91258.6 1258.6

1263.6 1679.1

ehv qtof 10021704 436 (15.395) Cm (436:441-408) 1: TOF MS ES+ 56629.8420.2214.1 405.2 1258.6 1259.6 1678.8 1679.0 1888.8

ehv qtof 10021704 435 (15.359) Cm (430:435-408) 1: TOF MS ES+ 56629.8420.2214.1 405.2

629.0627.7

630.3 1258.6 1259.61678.8

[5M+4H]4+

[4M+3H]3+

[6M+4H]4+

6 = range 460:465

5 = range 454:459

4 = range 448:453

3 = range 442:447

2 = range 436:441

1 = range 430:435

[M+H]+[M+2H]2+

[M+3H]3+

? ?

NC100717 (2.12 mg/ml 17feb10); FKAL 420-014; CV30

Time10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00

%

0

100

ehv qtof 10021704 3: Diode Array Range: 1.323e+115.21

UV

m/z 601.3

m/z 572.3

m/z 687.3

Conclusion

• One or possibile two compounds were found to elute at the rising edge of the main peak

• One compound was found to elute at the falling edge of the main peak

The use of PCA has become an essential tool within spectra interpretation of NIR, IR, RAMAN, UV of Fluorescence spectra.

The high number of wavelengths (~ 1200 wavelengths) makes PCA a necessary tool.

PCA is often used in combination with pre-spectral transformation as derivation, normalization and “scattering” corrections. All these tools are available in Unscrambler.

The PCA algorithm is VERY adequate to evaluate changes in DAD spectra as a function of retention time in LC-DAD

Principal Component Analysis on LC-DAD spectra – PCA Peak Purity

Principal Component Analysis on LC-DAD spectra – PCA Peak Purity

Why: To reveal any co-eluted constituents in LC interfering with the real purity.

How: Perform principal component analysis on UV/VIS DAD spectra from a LC run.

When:

•Impure (main) peak is suspected

•Evaluate resolution as a function of method conditions during method development to

•Control of GxP material and stored samples (at elevated temperatures)

•Compare new peaks with historical peaks

•Determine if a LC-MS analysis should be used / put up

Principal Component Analysis on LC-DAD spectra – PCA Peak Purity

How does PCA work:Data matrix300 nm 301 nm 302 nm 303 nm 304 nm 305 nm 306 nm

RT 1,01RT 1,02RT 1,03RT 1,04RT 1,05RT 1,06RT 1,07RT 1,08RT 1,09RT 1,10RT 1,11RT 1,12RT 1,13RT 1,14RT 1,15RT 1,16RT 1,17RT 1,18RT 1,19

The 3 most relevant chemometrics (statistical) parameters in peak purity PCA are:

•Loadings: Show which part of the UVVIS spectrum having largest change during retention time

•Scores: Show how the retention having the largest in the UVVIS spectra change (~Scores of PC1 is comparable to the single wavelength chromatogram at abs max)

•Residual variance: Show how the level of unexplained UVVIS variance at each time point

The first PC shows the largest change in spectra during time leaving noise behind. The second PC shows the second largest systematic change in the spectra leaving noise behind, and so on.

The score chromatogram from PC1 is usually shows the single wavelength chromatogram.The score chromatograms from PC2, PC3, PC4, etc., show chromatograms dependent on (small) changes in UVVIS spectrum that may indicate different chemical constituents than the main constituent.

Since the loadings from PC2, PC3, etc., is rest-variation after lower dimensionalities, these loadings spectra is not true spectra but show where the spectrum differ from main constituent spectrum.

The statistical parameters above are standard statistical output in Unscrambler and other chemometrics software.

PCA Peak-Purity

-300

0

300

600

900

12

14

16

18

20

RESULT3, PC(X-expl): 1(100%)

Samples

Scores

0

0.1

0.2

0.3

200 400 600 800

RESULT3, PC(X-expl): 1(100%)

X-variables

X-loadings

The score chromatogram from PC1 is a function of the largest variation in UVVIS spectra.

tR, min

Comparison of 650 nm abs max single wavelength chromatogram (upper) with score-chromatogram from PC1 (lower):

0

100

200

12

14

16

18

20

650

Samples

-300

0

300

600

900

12

14

16

18

20

RESULT3, PC(X-expl): 1(100%)

Samples

Scores

-3

0

3

6

9

12

14

16

18

20

650

Samples

-40

-20

0

20

12

14

16

18

20

RESULT3, PC(X-expl): 1(100%)

Samples

Scores

0

0.1

0.2

0.3

200 400 600 800

RESULT3, PC(X-expl): 1(100%)

X-variables

X-loadings

Sensitivity with PCA

The power of using PCA (noise reduction) on several wavelength compared to single wavelength in corresponding λ-area to get detailed information about purityConsequence: Small changes in UVVIS can be found by PCA

0

0.1

0.2

0.3

200 400 600 800

RESULT3, PC(X-expl): 1(100%)

X-variables

X-loadings

282nm

364nm

-0.2

0

0.2

0.4

12 14 16 18 20

282

Samples

Single 282nm (above) vs PCA on 252-300nm (below)

-0.1

0

0.1

0.2

12

14

16

18

20

364

Samples

Single 364nm (above) vs PCA on 354-398nm (below)

-0.6

-0.3

0

0.3

12

14

16

18

20

RESULT5, PC(X-expl): 1(100%)

Samples

Scores

-2

-1

0

1

12 14 16 18 20

RESULT4, PC(X-expl): 1(100%)

Samples

Scores

tR, min

tR, mintR, min

tR, min

PCA Peak-Purity – case study

Polymer based drug consisting of groups of different molecular weights

•High degree of co-elution / difficult (mission impossible?) to get peak separation

•Difficult to analyze with standard LC-MS equipment

•Challenging to achieve reproducible chromatograms

PCA Peak-Purity – case study

Detected by PCA

PCA Peak-Purity – case study – degraded sample

Evaluation of residual variance after PC1 and PC2 for this degraded sample revealed several components being important for selecting storage conditions.

No component here

Comparison with the LC-MS case

-60

-30

0

30

60

90

12.8

13.047

13.293

13.54

13.787

Samples

Scores

-0.2

-0.1

0

0.1

12.8

13.047

13.293

13.54

13.787

Samples

Scores

0

0.0005

0.0010

0.0015

12.8

13 13.2

13.4

13.6

13.8

Samples

X-variance Residual Sample Calibration Variance

-0.1

0

0.1

0.2

0.3

0.4

0.5

180 210 240 270 300

X-variables

X-loadings

UV Used for PCA

The PCA peak-purity indicated 2-3 peaks

2 peaks on the rising edge

1 broad “bump” peak on the falling edge

PC1 PC2

Residual variance after PC1

Pitfalls with PCA Peak-Purity

Non-linearity in absorbance can sometimes looks like co-eluting peaks. This have a often a systematic profile in residual variance.

Pitfalls with PCA Peak-Purity

MeCN gradient system will always affect the residual variance at λ < ~250 nm. Can be solved by (1) analyze a narrow time frame, (2) adjust to isocratic solvent composition over the specific peak or analyze DAD spectra above ~250 nm.

Pitfalls with PCA Peak-Purity

Evaluate the noise in scores, loadings and residual variance.

If the absorbance spectrum is suspected to be completely identical then LC-DAD is insufficient (~isomerism).

Noise profile

Thanks for your attention!