Analysis and Quantitation of Pharmaceutical Drugs using an Ultra High Capacity HPLC-chip

Martin Vollmer, Lukas Trojer, Stephan Buckenmaier

Agilent Technologies, Waldbronn, Germany

Chip design (Fig. 2) Superior Sensitivity and Limit of Quantitation

Introduction Ultra High Capacity Small Molecule Chip (UHC-Chip G4240-65002 A benchmark comparison between standard ESI and UHC-chip/MS
Agilent): was conducted. In both cases, the same QQQ and the same absolute
Analytical column (AC): 150 mm length, 75 x 50 m ID, Zorbax SB-C18, amount of sample (spiked in serum) was analyzed. With the chip a
Quantitation of drug metabolites in the early discovery 5 m, 80 50- 100 fold sensitivity increase was achieved (Fig 6 A). The overlay
phase is challenged by small sample volume and Enrichment column (EC): 500nl; 25mm length, 0.4 mm width, 0.05 mm of MS intensities (Fig. 6B) for both experiments clearly demonstrates
height these findings.
complex biological matrices. Efforts are immense to Structures: Multi-layer design: Laser ablated nanospray emitter, The absolute LOD in serum was 10 fg for atropine and imipramine
perform small animals studies and microdosing to microfluidic channels, and inlet openings. (Fig 7) for the chip. Quantifier and qualifer were reliably detected at
reduce significantly costs and to enhance throughput. Packing: G4240-65002: Zorbax SB, 5um in AC and EC this concentration . In addition, the UHC chip demonstrated high
HPLC-chip/MS provides an easy to use tool to achieve Chip material: Polyimid robustness towards samples in biological matrices. Total lifetime for
superior sensitivity and overcomes limitations usually Filter layer: Carbon implanted polyimid filter for increased lifetime, chips exceeded always 200 injections.
encountered by Nano-HPLC/MS. Integration of tightness, and robustness
functional elements onto a polymeric chip prevents x1 0 2 + E IC M R M (** -> 8 5 .5 0 0 0 0 -8 6 .5 0 0 0 0 , 1 1 5 .5 0 0 0 0 -1 1 6 .5 0 0 0 0 ...) 1 p g _ 1 3 .d

Results 102
9

peak dispersion and clogging and results thus into

8 .5
Atropine
EIC Quantifiers
8
7 .5
Imipramine

increased detection sensitivity, enhanced robustness

7
070802\1pg_13
6 .5
logP 4.2
6

and prolonged column lifetime.

5 .5
5

3 6
4 .5

Standard ESI source

4
3 .5 Atenolol Metoprolol

1 4 3
2 .5 logP 0
Zorbax C18 (80A) 2.1 x 150mm
240L/min

In this study a novel Ultra-High Capacity Chip (UHC) for

2
2 1. Atenolol 1 .5
1

small molecule analysis is presented (G4240 65002). 6 2. Caffeine 0 .5

0

3. Atropine 0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 6 .5 7 7 .5 8 8 .5 9 9 .5 10 1 0 .5 11 1 1 .5 12 1 2 .5 13 1 3 .5 14 1 4 .5 15

This chip enables highly sensitive analysis of a wide 104

Co u n ts v s. A c q u isitio n T ime (min )

A 4. Metoprolol
5. Propranolol
B x10 4
9
+ EIC MRM (** -> 85.50000-86.50000, 115.50000-116.50000 ...) 1pg_13.d

5 8.5

polarity range of compounds combined with the 6. Imipramine

8
7.5

acquisition of quantitative data in a routine DMPK 5 4 7

6.5

UHC-small molecule chip

6

environment. In addition, preliminary experiments are

5.5
5 Zorbax C18 (80A) 75m x 150mm
4.5 0.3L/min

presented employing a HILIC prototype chip for the

4
3.5
3

analysis of highly polar compounds which are difficult

3 2.5
2

to analyse with RP chromatography.

1.5
1
0.5
2 0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15
1 Counts vs. Acquisition Time (min)

Fig. 6. Comparison of a 4 compound drug mixture between Standard

Chip system and design ESI (A) and the UHC chip (B) at 1 pg.
Fig. 3. Analysis of a typical pharmaceutical drug test mixture (log P B shows an overlay of UHC-chip (blue) and Standard ESI (red). The
range 0 to 4.0). A: UHC-chip, 500nl EC B: Standard chip (40 nl EC). same absolute amount was injected in both experiments.
Quantifier Quantifier/Qualifier overlay Quantifier
Analysis of a Wide Polarity Range of Compounds Quantifier/Qualifier overlay

For DMPK and Metabolite ID a wide range of analytes from hydrophobic

+ MRM (281.2 -> 58.0) 10fg_300nl_2.d 281.2 -> 58.0 , 281.2 -> 86.0 + MRM (290.1 -> 124.1) 10fg_300nl_2.d 290.1 -> 124.1 , 290.1 -> 93.0

Counts

Counts

Counts

Counts
x10 2 x10 2 Ratio=236.9 x10 2 x10 2 Ratio=23.3
Imipramine
8 3.6 Atropine 3.6
3.4

to very hydrophlic needs to be detected and quantified.

7.5 3.4 3.4
3.2
3.2 3.2
3 7

2.8 A 6.5
A 3 3

In RP separation hydrophilic compounds are un-retained and elute in the 2.6 6

2.8
2.6
B 2.8
2.6
B
2.4 5.5
2.4 2.4

void volume. Nano-HPLC and HPLC-chip/MS require pre-concentration

2.2 5
2.2 2.2
2 4.5 2 2
1.8 1.8
4 1.8

due to the unfavorable ratio of injection volume to column volume.

1.6 1.6
3.5 1.6
1.4 1.4 1.4
3
1.2 1.2

During this step hydrophilic metabolites are frequently lost. With the
1.2
2.5
1 1 1
2
0.8 0.8 0.8
1.5

500nl enrichment column of the UHC chip it was possible to retain

0.6 0.6 0.6
0.4 1 0.4 0.4
0.2 0.5 0.2 0.2

quantitatively hydrophilic compounds to a log P value of 0 (Fig. 3 A)

0 0 0 0
10.4 10.6 10.8 11 11.2 11.4 11.6 10.4 10.6 10.8 11 11.2 11.4 11.6 8 8.2 8.4 8.6 8.8 9 9.2 8 8.2 8.4 8.6 8.8 9 9.2
Acquisition Time (min) Acquisition Time (min) Acquisition Time (min) Acquisition Time (min)

while in contrast with a standard chip hydrophilic analytes got partially

Fig. 1. HPLC-Chip/MS setup for quantitative small molecule
analysis. The Agilent HPLC-chip MS system consists of a 1200
Nano HPLC system, the HPLC-chip interface and the Agilent
G6410 Triple quadrupole.
lost (Fig. 3 B). Quantitation using the 6410 QQQ and the UHC chip was Fig. 7. Limit of quantitation. Quantitation of Imipramine (A) and
linear over a wide range of injection volumes (Fig. 4 A). Only for atenolol Atropine (B) in human serum. MRMs were recorded at 10 fg on
(log P 0) loss of analyte was observed exceeding 3 ul injection volume column using the UHC-Chip in combination with the G6410 QQQ and
(Fig. 4 B). the UHC chip.
6l
Analysis of a highly polar Analytes by HILIC
Atropine - 6 Levels, 6 Levels Used, 12 Points, 12 Points Used, 0 QCs
R esp o n se s

x10 6

R^2 = 0.9905
2.8

2.6
5l
Sample IN
2.4

2.2

A 4l
Metabolite ID and DMPK require often the analysis of highly polar
analytes which are not retained by reversed phase chromatography.
2

3l
1.8

Hydrophilic Interaction Chromatography (HILIC) is frequently applied

1.6

A5 A4
Enrichment Column Spray Tip 1pg/L
1.4

1.2 2l >20pg abs. enriched

500nl
for this purpose. This technique also simplifies workflows since
1

Injection-V: 1l 0.8

organic supernatants of precipitated serum samples can directly be

A6 A3 0.6

0.4

Waste
injected onto HILIC phases. A HILIC protoype chip was applied to
0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4

Separation Column
Concentration (ng/ml)

ID: 50 x 75 um, L: 150 mm

5 Atenolol - 6 Levels, 3 Levels Used, 12 Points, 7 Points Used, 0 QCs analyze a mixture of hydrophilic pharmaceuticals and vitamins (Fig 8).
R e sp o n se s

x10
A1 A2
2.2 R^2 = 0.9957
2.1

B
2

1.9

Analytical Pump 1.8

1.7

1.6

1.5

Separation Column 150mm dia 75m 1.4

1.3
Even up to 3L for the logP 0 compound
1.2

Enrichment Column 1.1

500nL
1

0.9

0.8

0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4
Concentration (ng/ml)
Sample IN
A5 A4

Fig. 4. Pre-concentration with different loading volumes. Loading

A6
Waste A3
A1 A2

volumes (from 1l to 6 l) with same absolute concentration on column

Analytical Pump
are applied on the UHC-chip EC. Atropine (log P 1.8) enriched
quantitatively for all loading volumes (A). For Atenolol (logP 0) linearity
is achieved up to 3 l injection volume (B).

Fig. 2. Chip design of the UHC small molecule chip, containing x10 4 + EIC MRM (** -> 85.50000-86.50000, 115.50000-116.50000 ...) 1000fg_16.d
9

a 500 nl enrichment column and a 150 mm separation column. 8.5

8 Atropine
0.08%
The chip is a multi layered polyimid device. Sample is loaded at 7.5
7
3.30% Imipramine Fig. 8. Analysis of hydrophilic compounds using a HILIC-HPLC-chip.
high flow on the enrichment column (4 l/min). Analysis is 6.5
6
0.07% Atropine (log P 2.2), atenolol (logP 0) thiamine (log P -2.1) and
performed at 300-600nl/min. 5.5
5 8.10% panthothenic acid (log P -1.3) were injected onto a HILIC prototype
070821\1000fg_10-20 chip packed with Zorbax Rx-Sil. EC: 500nl, AC: 150mm length, 50 x 75
4.5
4

m ID
3.5

Methods
3 Metoprolol
2.5 Atenolol
2 0.21% 0.10% RSD RT
1.5
4.10% 5.00% RSD Area
1
0.5

Instrumentation Conclusions
0
5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5

- Agilent 1200 HPLC chip/MS system (Fig. 1)

Counts vs. Acquisition Time (min)

- Agilent G6410 Triple Quadrupole Fig. 5. Repeatability of RT and area using the UHC-chip and 6410 Triple
Typical settings: Quadrupole. Displayed are 10 consecutive runs as overlays. UHC-small molecule Chip: Analysis of pharmaceutical
- Cap voltage 1850 V, 4l nitrogen drying gas, 300 C. compounds in bio-matrices over a wide polarity
Repeatability and precision of retention times and
- Fragmentation voltage and collision energy in MRM range
optimized for all transitions individually quantitation
Sensitivity Increase: 50-100 x vs. standard ESI
Mobile phases: 0.1 % Formic acid (A)
Acetonitrile , 0.1% Formic acid (B) A compound mixture of 4 pharmaceutical standards was spiked into a LOD (abs): 10 fg for atropine and imipramine in serum.
Gradients: typically from 2% to 70% B for RP and from 100% human serum sample and run to run reproducibility for retention times
and area was investigated in MRM mode. With the UHC-chip excellent UHC-chip suited for routine operation, increased
to 20 % for HILIC. robustness and lifetime to analyze small sample
Flow rates: 4 l/min sample loading retention time reproducibility with relative standard deviations between
0.07% and 0.2% (Fig. 5) were achieved. For quantitation RSDs in the quantities.
300nl/min separation
range of 3-8% were obtained which is significantly below the 20% range
that is required by FDA.