Fluorescence detection in liquid chromatography

A new approach to lower limits of detection and easy spectral analysis

A primer

A new approach to lower limits of detection and easy spectral analysis

Applications of fluorescence detection in liquid chromatography

Rainer Schuster and Helmut Schulenberg-Schell

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Copyright 2000 Agilent Technologies All rights reserved. Reproduction, adaption, or translation without proir written permission is prohibited, except as allowed under the copyright laws. Printed in Germany 01/00 Publication number 5968-9346E

Acknowledgment

The authors would like to thank our Agilent Technologies colleagues Thomas Drr, Angelika Gratzfeld-Huesgen and Ludwig Huber for fruitful discussions and review of the manuscript. Udo Huber and Angelika Gratzfeld-Huesgen contributed to the applications.

Preface

High performance liquid chromatography (HPLC) is the method of choice for separation and quantitation of polar and nonvolatile compounds. Retention time is the main tool for identification of analytes. The complexity of real world analytical problems requires the confirmation of peak identity through additional qualitative information. This can be provided from several detector types: diode array, mass spectrometric or fluorescence detectors. In 1982 HPs chemical analysis group (now part of Agilent Technologies) introduced the first commercially available diode array detector (DAD) for HPLC. This detector added a third dimensionwavelengthto the chromatogram in addition to retention time and signal intensity. The diode array detector records UV/Visible absorption spectra in milliseconds while compounds are eluting from a column. A single chromatographic run provides quantitative results from the signal intensity and qualitative spectral information for peak confirmation. The improvements in detector design and the lower price have made it a sensitive and cost-effective tool that is about to replace single wavelength UV-detectors, even those performing routine analyses. Since the mid 1990s, mass spectrometric detectors for liquid chromatography (LC-MSD) have been available to provide analysts with both molecular weight and structural information for peak confirmation. The ruggedness and easy use of the instrumentation make this technique available for the chromatographer. From research and development, the technique will make its way into the cost-sensitive routine QA/QC laboratories. The fluorescence detector (FLD) is one of the most sensitive detectors in liquid chromatography. Both excitation and emission fluorescence spectra help to characterize individual compounds. While excitation spectra are identical to UV/Visible absorption spectra, emission spectra can give additional information. Until recently, however, FLDs have been built to provide single-wavelength information.

Fluorescence spectra had to be acquired under stop-flow conditions and data analysis was time-consuming and cumbersome compared to data analysis of UV/Visible spectra. A completely new approach to fluorescence detector design has overcome these drawbacks and even improved the sensitivity. The Agilent 1100 Series fluorescence detector acquires spectra online simultaneously with the detector signal. During method development, one or two chromatographic runs can be sufficient to optimize wavelength settings for a series of analytes. This replaces tedious stopflow experiments for each individual compound. Fluorescence spectral data are analyzed with the same easy-to-use software tools as diode array spectra. In addition, up to four wavelengths can be recorded simultaneously to replace timetable-based wavelength switching. This ensures maximum sensitivity and selectivity without sacrificing the reliability of the analytical method in routine analysis of real-world samples. This primer introduces the new fluorescence detector technology and describes new strategies for rapid method development. A selection of applications demonstrates the wealth of information available with this new approach to fluorescence detection.

Table of content
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Chapter 1 Fluorescence detector technology ..................................................................................... 9

How the fluorescence detector works .......................................................................................... 11 Data handling ................................................................................................................................... 12 How to measure limits of detection .............................................................................................. 14

Chapter 2 Strategies for rapid method development ....................................................................... 15

Step 1: Check the HPLC system for impurities ............................................................................ 17 Step 2: Optimize limits of detection and selectivity ................................................................... 18 Procedure I Take a fluorescence scan .................................................................................... 19 Procedure II Take two HPLC runs with FLD ......................................................................... 21 Procedure III Make a single run with the Agilent 1100 Series DAD/FLD combination ... 23 Step 3: Set up routine methods ...................................................................................................... 25 Multi wavelength detection ........................................................................................................ 25 Fluorescence spectral libraries for peak confirmation ........................................................... 26

Chapter 3 The applications ................................................................................................................... 29

Environmental: Polynuclear aromatic hydrocarbons ......................................................................................... 30 Carbamates ................................................................................................................................... 33 Glyphosate .................................................................................................................................... 37 Food: Mycotoxins ................................................................................................................................... 40 Aflatoxins B1/B2 and G1/G2 ....................................................................................................... 40 Ochratoxin A ................................................................................................................................ 42 Vitamins B2 and B6 ...................................................................................................................... 43 Pharmaceutical: Quinidine....................................................................................................................................... 46 Warfarin ......................................................................................................................................... 48 Amino acids .................................................................................................................................. 50 References........................................................................................................................................ 52 Index ................................................................................................................................................. 53

Chapter 1

Fluorescence detector technology

Fluorescence detector technology

Fluorescence detectors offer high selectivity combined with superior limits of detection (LOD) compared to UV detectors. Only about 10 percent of organic molecules have fluorophore structures, which enable the molecules to absorb light over a range of wavelengths. This takes electrons in the molecule to an excitation level as with any other molecule containing a chromophore. The fluorescent molecule has the ability to emit the absorbed energy at longer wavelengths (ref. 1). If the fluorescent light intensities are recorded while the excitation wavelength is changed and the emission wavelength is fixed, an excitation spectrum can be obtained. Or the excitation wavelength may be fixed and the emission wavelength changed. This procedure provides an emission spectrum of the analyte. Compounds without this native fluorescence may be derivatized to attach a fluorescent marker molecule in a pre- or post-column reaction (ref. 2). Fluorescence detectors offer limits of detection down to the ppt level. The signal intensities are very low compared to UV absorption and they are measured ideally versus a very low background noise level. This is inherently more sensitive than comparing two relatively large signals from a blank and a sample as done in UV absorption spectroscopy. However, the sensitivity of fluorescence detection is dependent on both the fluorophore properties and the detector design and settings. The response of a fluorophor is characterized by molar absorptivity and quantum yield at the applied experimental conditions. The sensitivity of the fluorescence detector depends on several factors: source intensity, efficiency of the optical system, bandwidth and so forth.

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How the fluorescence detector works

Previous fluorescence detectors were equipped with motordriven gratings for programmable excitation and emission wavelengths. The detector layout enabled positioning for a single wavelength at a time and required stop-flow conditions for acquisition of spectra. Thus, acquiring fluorescence spectra was time-consuming and optimizing excitation and emission wavelengths was not possible without disturbing chromatographic separations. The new optical design of the Agilent 1100 Series fluorescence detector is illustrated in figure 1. A Xenon flash lamp is used to offer the highest light intensities for excitation in the UV range. The flash lamp ignites only for microseconds to provide light energy. Each flash causes fluorescence in the flow cell and generates an individual data point for the chromatogram. Since the lamp is not powered on during most of the detector operating time, it offers a lifetime of several thousand hours. No warmup time is needed to get a stable baseline. A holographic grating is used as a monochromator to disperse the polychromatic light of the Xenon lamp. The desired wavelength is then focused on the flow cell for optimum excitation. To minimize stray light from the excitation side of the detector, the optics are configured such that the emitted light is recorded at a 90 degree angle to the incident light beam. Another holographic grating is used as the emission monochromator. Both monochromators have optimized light throughput in the visible range. A photomultiplier tube is the optimum choice to measure the low light intensity of the emitted fluorescence light. Since flash lamps have inherent fluctuations with respect to flash-to-flash intensity, a reference system based on a photodiode measures the intensity of the excitation and triggers a compensation of the detector signal. Since the vast majority of emission maxima are above 280 nm, a cut-off filter (not shown) prevents stray light below this wavelength to enter the light path to the emission monochromator. The fixed cut-off filter and bandwidth (20 nm) avoid the hardware checks and documentation

Emission monochromator

Xenon flash lamp

Lens Mirror Lens Photomultiplier

Excitation monochromator

Sample

Photodiode

Figure 1 Optical design of the Agilent 1100 Series fluorescence detector

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Fluorescence detector technology

Fluorescence detector technology

work that is involved with an instrument that has exchangeable filters and slits. The excitation and emission monochromators can switch between signal and spectral mode. In signal mode they are moved to specific positions that encode for the desired wavelengths, as with a traditional detector. This mode offers the lowest limits of detection since all data points are generated at a single excitation and emission wavelength. The spectral mode is used to obtain multi-signal or spectral information. The ignition of the flash lamp is synchronized with the rotation of the gratings, either the excitation or emission monochromator. The motor technology for the gratings is a long-life design as commonly used in highspeed PC disk drive hardware. Whenever the grating has reached the correct position during a revolution, the Xenon lamp is ignited to send a flash. The flash duration is below two microseconds while the revolution of the grating takes less than 14 milliseconds. Because of the rotating monochromators, the loss in sensitivity in the spectral mode is much lower compared to conventional dual-wavelength detection with UV detectors. In addition to its use as a detector for liquid chromatography, the Agilent 1100 Series FLD offers capabilities for offline measurements in a refillable cuvette to obtain excitation and emission spectra in a single task for a pure compound. The result of this fluorescence scan can be viewed in a three-dimensional plot showing excitation wavelength, emission wavelength and fluorescence intensity on the axis.

Data handling

The sequence of data handling is shown schematically in figure 2. The example describes the collection of excitation spectra and a signal with Ex 250 nm/Em 350 nm. Alternating flashes are used either for chromatographic signals or to contribute to spectra acquisition. About every 14 millisec-

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onds, a data point is obtained. The spectrum starts at 230 nm. Just as with a DAD system, the fluorescence spectra and signals can be watched online and both types of data are stored on the PC hard disk. Additional signals can be extracted from the spectral data set during post-run data analysis. Spectral data points are corrected automatically for intensity changes over time based on the signal data.

Chromatogram Flash# Time Ex Em Intensity [msec] [nm] [nm] 1* 0 250 350 5 2 14 230 350 4 3 28 250 350 8 4 42 240 350 15 5 56 250 350 15 6 70 250 350 30 7 84 250 350 30 8 98 260 350 25 *flash duration = 1 microsecond

Time Spectrum

Figure 2 Schematic representation of data flow in spectral mode One signal at Ex 250 nm/Em 350 nm is recorded and excitation spectra are taken starting at Ex 230 nm.

Wavelength

With chromatographic data in three dimensions (emission or excitation wavelengths, retention time and intensity), the analysis can be displayed either in a three-dimensional plot or in a two-dimensional isofluorescence plot. Different colors signify different fluorescence intensities. Spectra can be evaluated against spectra from customized spectral libraries to determine compound identity or to control peak purity within a peak. While the quality of fluorescence spectra has proven to be useful for several applications, it has to be emphasized that UV/Vis spectra obtained with a diode array detector are of superior quality, especially for trace analysis.

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Fluorescence detector technology

Fluorescence detector technology

How to measure limits of detection

Fluorescence spectrophotometers can be qualified by the signal-to-noise ratio (S/N) for the Raman band for water or as a limit of detection (LOD) for a specific fluorescent compound.(ref.3) The Raman band is a result of Raman light scattering and not due to fluorescence. It simulates the phenomenon of fluorescence as it involves an initial light signal at a specific wavelength that causes a signal to occur at a longer wavelength. The specification based on Raman stray light is given as the ratio of signal to noise with the excitation wavelength at 350 nm and emission at 397 nm. The short-term noise is measured at 397 nm (figure 3) according to the procedures described in ASTM method 1657/94. For both values, the dark value at 450 nm (where no stray light appears) is taken as a reference point for the scale. Raman values greater than 400 are typical for fluorescence detectors in HPLC. If anthracene is used to measure detector specifications, limits of detection as low as 10 femtogram anthracene are possible.

Figure 3 Raman signal-to-noise (S/N) measurement Signal and ASTM noise at 397 nm (water), excitation at 350 nm, PMT=12, response time 8 s

LU 38 36 34 32 30 28 26 24 0 2.5 5

Signal and ASTM noise at 350/397 nm

Raman S/N =

Signal (397 nm) dark value (450 nm) ASTM noise (397 nm) dark value (450 nm)

Dark value at 350/450 nm

7.5

10

12.5

15

17.5

20

[min]

14

Chapter 2

Strategies for rapid method development

15

Method development

Fluorescence detectors are used in liquid chromatography when superior limits of detection and selectivity are required. Thorough method development, including spectra acquisition, is fundamental to achieve good results. This chapter describes three different steps that can be taken with the Agilent 1100 Series fluorescence detector. Table 1 gives an overview of how to benefit from the operation modes during these steps.

Step 1: Check system

Step 2: Optimize limits of detection and selectivity Determine simultaneously the excitation and emission spectra of a pure compound Perform wavelength switching Determine Ex/Em spectra for all separated compounds in a single run Activate up to four wavelengths simultaneously

Step 3: Set up routine methods

Fluorescence scan

Find impurities (for example, in solvents and reagents)

Signal mode

Use for lowest limits of detection Collect online spectra, perform library search, determine peak purity Replace wavelength switching

Spectral mode/ multi-wavelength detection

Table 1 Steps for thorough method development

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Step 1: Check the HPLC system for impurities

A critical issue in trace level fluorescence detection is to have an HPLC system free of fluorescent contamination. Most contaminants derive from impure solvents. Taking a fluorescence scan is a convenient way to check the quality of the solvent in a few minutes. This can be done, for example, by filling the FLD cuvette directly with the solvent for an offline measurement even before the start of a chromatographic run. The result can be displayed as an isofluorescence plot or a three-dimensional plot. Different colors reflect different intensities. Figure 4 shows a sample of slightly impure water which was planned for use as mobile phase. The area where fluorescence of the contaminated water sample can be seen is between the stray light areas: the first- and second-order Raleigh stray light and Raman stray light. Since excitation and emission wavelength are the same for Raleigh stray light, the area of first-order Raleigh stray light is visible in the left upper area of the diagram. The Raman bands of water are seen below the first-order Raleigh stray light. Since the cut-off filter cuts off light below 280 nm, the second-order Raleigh stray light starts above 560 nm.
Impurity 450 nm 1. order Raman 2. order

Figure 4 Isofluorescence plot of a mobile phase A pure water sample was put into the flow cell. Spectra were recorded at 5 nm step sizes.

220 nm Emission 600 nm

Excitation

Stray light acts in the same way as impurities in that it simulates background noise. In both cases, a higher noise level and therefore a higher limit of detection are obtained. This indicates that high sensitivity measurements should be done away from wavelength settings that have a high stray light background. 17

Method development

Method development

Step 2: Optimize limits of detection and selectivity

To achieve optimum limits of detection and selectivity, analysts must find out about the fluorescent properties of the compounds of interest. Excitation and emission wavelengths can be selected for optimum limits of detection and best selectivity. In general, fluorescence spectra obtained with different instruments may show significant differences depending on the hardware and software used. (ref. 4) The traditional approach is to extract an appropriate excitation wavelength from the UV spectrum that is similar to the fluorescence excitation spectrum (see figure 5) and to record the emission spectrum. Then with an optimum emission wavelength determined, the excitation spectrum is acquired. These tasks have to be repeated for each compound using either a fluorescence spectrophotometer or stop-flow conditions in HPLC. Usually each compound requires a separate run. As a result, a set of excitation and emission spectrum is obtained (figure 5) for each compound. Since this is a tedious procedure, it is applicable only when there is a limited number of compounds of interest.
Norm. 40 35 30 25 20 15
Excitation Emission

Figure 5 Excitation and emission spectra of quinidine Excitation spectrum with emission at 440 nm, emission spectrum with excitation at 250 nm of 1 g/ml quinidine. Detector settings: step size 5 nm, PMT 12, Response time 4 s.

10 5 0 250 300 350 400 450 500 550 600

Wavelength [nm]

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The Agilent 1100 Series HPLC offers three different ways to obtain complete information on a compounds fluorescence: Procedure I - Take a fluorescence scan offline for a single compound as described above for the mobile phase. This is done preferably with a manual FLD cuvette when pure compounds are available. Procedure II - Use two HPLC runs with the Agilent 1100 Series FLD to separate the compound mix under known conditions and acquire emission and excitation spectra separately. Procedure III - Use an Agilent 1100 Series FLD/DAD combination and acquire UV/Visible spectra (equivalent to excitation spectra) with the DAD and emission spectra with the FLDboth in a single run.

Procedure I Take a fluorescence scan

Because fluorescence spectra traditionally have not been easily available with previous HPLC fluorescence detectors, standard fluorescence spectrophotometers have been used in the past to acquire spectral information for unknown compounds. Unfortunately this approach limits optimization, as there are differences expected in optical design between an HPLC detector and a dedicated fluorescence spectrophotometer, or even between detectors. These differences can lead to variations for the optimum excitation and emission wavelengths. The Agilent 1100 Series fluorescence detector offers a fluorescence scan that delivers all spectral information previously obtained with a standard fluorescence spectro-

19

Method development

Method development

photometer, independent of the HPLC fluorescence detector. Figure 6 shows the complete information for quinidine as obtained with the Agilent 1100 Series FLD and a manual cuvette in a single offline measurement. The optima for excitation and emission wavelengths can be extracted as coordinates of the maxima in the three dimensional plot. One of the three maxima in the center of the plot can be chosen to define the excitation wavelength. The selection depends on the additional compounds that are going to be analyzed in the chromatographic run and the background noise that may be different upon excitation at 250 nm, 315 nm or 350 nm. The maximum of emission is observed at 440 nm.

straylight 1. order

350 nm Ex 315 nm Ex

250 nm Ex

Figure 6 Characterization of a pure compound from a fluorescence scan All excitation and emission spectra of Quinidine (1 g/ml) are shown in one graphic. Fluorescence intensity is plotted vs. excitation and emission wavelengths. Figure 5 gives detector settings. Ex-axis Em-axis 440 Em

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Procedure II Take two HPLC runs with the FLD

The conditions for the separation of organic compounds such as polyaromatic nuclear hydrocarbons (PNAs) are well described in various standard methods, including commonly used EPA and DIN methods. Achieving the best detection levels requires checking for the optimum excitation and emission wavelengths for all compounds. Yet taking fluorescence scans individually makes this a tedious process. A better approach is to acquire spectra online for all compounds during a run. This speeds up method development tremendously. Two runs are sufficient for optimization. During the first run, one wavelength is chosen in the low UV range for the excitation wavelength and a spectral range for the emission wavelength. Most fluorophores show strong absorption at these wavelengths. Excitation is sufficient for collecting emission spectra. Figure 7 contains all emission spectra obtained in a single run from a mix of 15 PNAs. This set of spectra is used to set up a timetable for optimum emission wavelengths for all compounds. The individual compound spectra in the isofluorescence plot show that at least three emission wavelengths are needed to detect all 15 PNAs properly:
0 min: 350 nm 8.2 min: 420 nm 19.0 min: 500 nm for naphthalene to phenanthrene for anthracene to benzo(g,h,I)perylene for indeno(1,2,3-cd)pyrene

In the second run, three setpoints for emission wavelengths are entered into the time-program and excitation spectra are recorded, as shown in figure 8. The area of high intensity (red) is caused by stray light when emission spectra overlap with the excitation wavelength. This can be avoided by fitting the spectral range automatically. Excitation at 260 nm is most appropriate for all PNAs. 21

Method development

Method development

Conditions for figure 7 and 8 Column Vydac, 2.1 200 nm, PNA, 5 m Mobile phase A = water; B = acetonitrile Gradient 3 min, 60 %B; 14 min, 90 %B; 22 min, 100 %B Flow rate 0.4 ml/min Column temperature 18 C Injection volume 5 l FLD settings PMT 12, response time 4 s, step size 5 nm Figure 7 Optimization of the time-program for the emission wavelength This shows the isofluorescence plot of emission spectra for 15 PNAs (5 g/ml) with a fixed excitation wavelength (260 nm).

1 2 3 4 5 6 7

Naphthalene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene

8 9 10 11 12 13 14

Benz(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benz(a)pyrene Benzo(g,h,i)perylene Indeno(1,2,3-cd)pyrene 11 12 5 8 10 6 7 9 14 13

LU 60 50 40 30 20 10 0
600 nm

2 1 3 4

2.5

7.5

10

12.5

15

17.5

22.5 20 Time [min]

Em-spectra

300 nm

fixed Ex

1 2 3 4 5 6 7

Naphthalene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene

8 9 10 11 12 13 14

Benz(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benz(a)pyrene Benzo(g,h,i)perylene Indeno(1,2,3-cd)pyrene 11

LU 60 50 40 30 20 10 0
400 nm

12 2 1 3 4 5 6 7 8 10 9 13 14

2.5

7.5

10

12.5

15

17.5

20 22.5 Time [min]

Excitation spectra

Figure 8 Optimization of the time-program for the excitation wavelength

220 nm 350 nm 420 nm 500 nm Emission switching

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Time [min] 0 8.2 19.0

Excitation wavelength [nm] 260 260 260

Emission wavelength [nm] 350 420 500

The obtained data are combined to setup the time-table for for best limit of detection and selectivity. The optimized switching events for this example are summarized in table 2.

Table 2 Timetable for the analysis of 15 polynuclear aromatic hydrocarbons This timetable gives the conditions for optimum detection based on the results of two chromatographic runs.

Procedure III Make a single run with the Agilent 1100 Series DAD/FLD combination
For most organic compounds, UV-spectra from diode array detectors are nearly identical to fluorescence excitation spectra. Spectral differences are caused by specific detector characteristics such as spectral resolution or light sources. In practice, combining a diode array detector with a fluorescence detector in series gives the full data set needed to achieve the optimum fluorescence excitation and emission wavelengths for a series of compounds in a single run. With the UV/Visible/excitation spectra available from the diode array detector, the fluorescence detector is set to acquire emission spectra with a fixed excitation wavelength in the low UV range. The example is taken from the quality control of carbamates. Samples are analyzed for the impurities 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP). Reference samples of DAP and AHP were analyzed with diode array and fluorescence detection. Figure 9 shows the spectra obtained from both detectors for DAP. The excitation spectrum of DAP is very similar to the UV absorption spectrum from the diode array detector. Figure 10 shows the successful application of the method to a carbamate sample and a pure mixture of DAP and AHP for reference. The column was overloaded with the non-fluorescent carbamate (2-benzimidazole carbamic acid methylester/ MBC) to see the known impurities, AHP and DAP.

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Method development

Method development

Conditions Column

Norm. 265 nm 35 30 25 20 15 10 5 0 200 250 300 350 400 450 500 550 Excitation DAD-spectra Emission UV 430 nm

Zorbax SB, 50 2 mm, PNA, 5 m Mobile phase A = water; B = acetonitrile Gradient 0 min, 5 %B; 10 min, 15 %B; Flow rate 0.4 ml/min Column temperature 35 C Injection volume 5 l FLD settings PMT 12, response time 4 s, step size 5 nm, Ex 265 nm and 430 nm, Em 540 nm

Wavelength [nm]
Figure 9 UV-spectrum and fluorescence spectra for 2,3-diaminophenazine (DAP) This is an impurity of carbamates. The excitation spectrum in a second run shows the similiarity of UV-spectra and fluorescence excitation spectra. An excitation wavelength at 265 nm was used for taking the emission spectrum and an emission wavelength at 540 nm was used for taking the excitation spectrum.

2-amino-3-OH-phenazine

LU 0.8 0.6

Unknown MBC

2,3-diaminophenazine

265/540 nm 0.4 430/540 nm

Figure 10 Qualitative analysis of MBC (2-benzimidazole carbamic acid methylester) and impurities The two upper traces are obtained using two different excitation wavelengths. The lower trace is a pure standard of the known impurities.

0.2 0 0 2 4 6 8 Standard 10 12 Time [min]

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Step 3: Set up routine methods

In routine analysis, sample matrices can have a significant influence on retention times. For reliable results, sample preparation must be thorough to avoid interferences or HPLC methods must be rugged enough. With difficult matrices, simultaneous multi-wavelength detection offers more reliability than timetable-controlled wavelength switching. The Agilent 1100 Series FLD can, in addition, acquire fluorescence spectra while it records the detector signals for quantitative analysis. Therefore qualitative data are available for peak confirmation and purity checks in routine analysis.

Multi wavelength detection

Time-programmed wavelength switching traditionally is used to achieve low limits of detection and high selectivity in routine quantitative analysis. Such switching is difficult if compounds elute closely and require a change in excitation or emission wavelength. Peaks can be distorted and quantitation made impossible if wavelength switching occurs during the elution of a compound. Very often this happens with complex matrices, influencing the retention of compounds. In spectral mode, the Agilent 1100 Series FLD can acquire up to four different signals simultaneously. All of them can be used for quantitative analysis. Apart from complex matrices, this is advantageous when watching for impurities at additional wavelengths. It is also advantageous for reaching low limits of detection or increasing selectivity through optimum wavelength settings at any time. The number of data points acquired per signal is reduced and thus limits of detection may be higher, depending on the detector settings compared to the signal mode. PNA analysis, for example, can be performed with simultaneous multi wavelength detection instead of wavelengthswitching. With four different wavelengths for emission, all 15 PNAs can be monitored (figure 11). 25

Method development

Method development

Vydac, 2.1 250 mm, PNA, 5 m Mobile phase A = water; B = acetonitrile Gradient 3 min, 60 %B; 14.5 min, 90 %B; 22.5 min, 95 %B Flow rate 0.4 ml/min Column temperature 22 C Injection volume 2 l FLD settings PMT 12, response time 4 s

Conditions Column

1 excitation WL at 260 nm 4 emission WL at 350, 420, 440 and 500 nm

LU 180 160 140 120 100 80

1 2 3 4 Ex=275, Em=350, TT Reference chromatogram with switching events 5

1 2 3 4 5 6 7

Naphthalene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene

8 9 10 11 12 13 14 15 10

Benz(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benz(a)pyrene Dibenzo(a,h)anthracene Benzo(g,h,i)perylene Indeno(1,2,3-cd)pyrene

6 11 12 7 8 9

15
13 14

Figure 11 Simultaneous multi wavelength detection for PNA-analysis The upper trace was received with traditional wavelength switching. Ex/Em = 260/420 nm Ex/Em = 270/440 nm Ex/Em = 260/420 nm Ex/Em = 290/430 nm Ex/Em = 250/550 nm

60 40 20 0

Ex=260, Em=500 Ex=260, Em=440 Ex=260, Em=420 Ex=260, Em=350

10

15

20

25 Time [min]

Fluorescence spectral libraries for peak confirmation

Previously, only diode array detectors and mass spectrometric detectors could deliver spectral information on-line to confirm peak identity as assigned by retention time. Now, fluorescence detectors provide an additional tool for automated peak confirmation and purity control. No additional run is necessary after the quantitative analysis. During method development, fluorescence excitation and emission spectra are collected from reference standards and entered into a libraryat the choice of the method developer. All spectral data from unknown samples can then be compared automatically with library data. Table 3 illustrates

26

this principle using a PNA analysis. The match factor given in the report for each peak indicates the degree of similarity between the reference spectrum and the spectra from a peak. A match factor of 1,000 means identical spectra. In addition, the purity of a peak can be investigated by comparing spectra obtained within a single peak. When a peak is calculated to be within the user-defined purity limits, the purity factor is the mean purity value of all spectra that are within the purity limits. The reliability of the purity and the match factor depends on the quality of spectra recorded. Because of the lower number of data points available with the fluorescence detector in general, the match factors and purity data obtained show stronger deviations compared to data from the diode array detector, even if the compounds are identical.

Meas. Library CalTbl RetTime Sig Amount Purity Library Name [min] [min] [min] [ng] Factor # Match 4.859 6.764 7.137 8.005 8.841 9.838 10.439 12.826 13.340 15.274 16.187 16.865 18.586 19.200 20.106 4.800 7.000 7.100 8.000 8.800 10.000 10.400 12.800 13.300 15.200 16.200 16.900 18.600 19.100 2 0.000 5.178 7.162 7.544 8.453 9.328 10.353 10.988 13.469 14.022 16.052 17.052 17.804 19.645 20.329 21.291 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.47986e-1 2.16156e-1 1.14864e-1 2.56635e-1 1.76064e-1 2.15360e-1 8.00754e-2 1.40764e-1 1.14082e-1 6.90434e-1 5.61791e-1 5.58070e-1 5.17430e-1 6.03334e-1 9.13648e-2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 993 998 995 969 993 997 1000 998 999 999 998 999 999 995 991 Naphthalene@em Acenaphthene@em Fluorene@em Phenanthrene@em Anthracene@em Fluoranthene@em Pyrene@em Benz(a)anthracene@em Chrysene@em Benzo(b)fluoranthene@em Benzo(k)fluoranthene@em Benz(a)pyrene@em Dibenz(a,h)anthracene@em Benzo(g,h,i)perylene@em Indeno(1,2,3-cd)pyrene@em

Table 3 Peak confirmation using a fluorescence spectral library This shows an automated library search based on the emission spectra from a PNA reference sample.

27

Method development

Method development

28

Chapter 3

The applications
Environmental Polynuclear aromatic hydrocarbons Carbamates Glyphosate Food Aflatoxins B1/B2 and G1/G2 Ochratoxine A Vitamins B2 and B6 Pharmaceutical Quinidine Warfarin Amino acids

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Environmental applications

Polynuclear aromatic hydrocarbons

Polynuclear aromatic hydrocarbons (PNA) are formed during pyrolysis or incomplete combustion in industrial or private heaters, automobile exhaust fumes and tobacco smoke. These compounds are in many oil products such as diesel fuel, gasoline and bitumen. PNAs have become a ubiquitous class of compounds found in all environmental matrices, including air, soil and water. Many PNAs have been found to be carcinogenic or mutagenic. Because their structures differ, some are more carcinogenic than others. Benzo(e)pyrene, for example, is a weak carcinogen, while isomeric benzo(a)pyrene is a strong carcinogen. For this reason, maximum concentration limits have been set for each individual PNA in air, soil and especially in water samples. Most environmental regulations in the U.S., many countries in Asia, and eastern Europe require PNA analysis according to U.S. Environmental Protection Agency (EPA) methods. These include analysis of 16 individual PNAs. In western Europe, most official methods describe the analysis of the same set of compounds as the EPA methodsexcept the European methods do not include acenaphthylene, a compound which does not show any fluorescence. A rapid method for analyzing only six PNAs using a fast isocratic HPLC method is described in the German standard method DIN 38 407 F8.

Experiments and results

With the rapid HPLC method, a PNA analysis is achieved in less then 10 min on a 25 cm microbore column (shown in figure 12). The lowest limits of detection for five of the six compounds is found at Ex 360 nm/Em 429 nm. For the last compound, the optimum is found at Ex 360/Em 490 nm and the compromise (Ex 360/Em 465 nm) gives medium limits

30

of detection for all. The detection limit for five of the PNAs is down to 100 fg and 1 pg absolute for indeno(1,2,3cd)pyrene (Ex 360 nm/Em 490 nm), the last eluting compound. Increasing the injection volume achieves even better detection limitsdown to low ppt levels.

Conditions Isocratic method Column Vydac, 2.1 250 nm, PNA, 5 m Mobile phase Water/acetonitrile = 10/90 Flow rate 0.4 ml/min Column temperature 18 C Injection volume 75 l FLD settings PMT 12, response time 4 s, step size 5 nm, Ex 360 nm, Em 465 nm

LU 10

LOD - 4.9ppt 1 2 3 4

1 2 3 4 5 6

Fluoranthene Benzo(k)fluoranthene Benzo(b)fluoranthene Benz(a)pyrene Benzo(ghi)perylene Indeno(1,2,3-cd)pyrene

6
LOD - 7.6ppt 5

4
6 LOD - 26ppt

75ul ( 2ppb) = 150 pg Position-mode

2
LOD - 3.3ppt

10

12

14

16

18 Time [min]

Figure 12 Determination of PNAs according to DIN 38407 F8 down to a low ppt-level A 2 ppb reference standard was analyzed at Ex 360 nm/Em 465 nm, with an injection volume of 75 l. The limits of detection (LOD) are given at the S/N ratio=2.

Most PNA standards contain the antioxidant trichlorophenol, which coelutes with chrysene. The antioxidant is so far observed only in the DAD analysis because of the UV absorption spectrum. With simultaneous multi wavelength detection, both chrysene at Ex 260/Em 420 nm and trichlorophenol at Ex 260/Em 310 nm can be quantitated selectively, as shown in figure 13. This is useful information when checking the stability of PNA standards.

31

Environmental applications

Environmental applications

Conditions Gradient method Column Vydac, 2.1 250 nm, PNA, 5 m Mobile phase A = water, B = acetonitrile Gradient 3 min, 50 % B, 14 min, 90 % B, 22 min, 100 % B, Flow rate 0.4 ml/min Column temperature 18 C Injection volume 5 l FLD settings PMT 12, response time 4 s, step size 5 nm, Ex=260 nm, Em=310 and 420 nm

LU 30 25 20 15 10 5 14 16

Trichlorophenol Ex=260, Em=310 Chrysene Ex=260, Em=420

18

20

22 Time [min]

Figure 13 Control of antioxidants in PNA reference standards with the Agilent 1100 Series fluorescence detector With multi wavelength detection, the antioxidant trichlorophenol and the PNAs could be analyzed in a single run, each at a specific wavelength.

Conclusions
Fluorescence detection is the most sensitive HPLC detector for PNA analysis. Detection limits are in the low ppt range. Simultaneous multi wavelength detection can replace time-programmed wavelength switching. Spectral mode fluorescence detection makes PNA methods more reliable, as it can provide information on additives and impurities not seen in single-wavelength detection.

32

Carbamates

Pesticides are regarded as essential to protect the quality of food during production, storage and distribution. The persistence of these chemicals requires monitoring of all major pesticides in crops as well as the environment. Carbamates are mainly used as insecticides on fruits and vegetables. According to EPA method 531.1, carbamates like the herbicide glyphosate need a postcolumn derivatization step. To meet the specified limits of detection, a fluorophor is attached to compounds separated on a dedicated column. Ortho-Phthalaldehyde is the reagent used most frequently.

Experiments and results

Carbamates are separated on dedicated columns. After hydrolysis of the compounds in the effluent, the derived methylamines react with an o-Phthalaldehyde/Thiofluor solution to the corresponding isoindole.

Pickering Carbamate column, 150 4.6 nm, Mobile phase A = water, B = methanol Gradient 0 min, 15 % B, 29 min, 100 % B, Flow rate 1.0 ml/min Column temperature 42 C Injection volume 1 l FLD settings PMT 12, response time 4 s, step size 5 nm, Post-column conditions in the Pickering system: OPA reagent for derivatization, reactor for hydrolysis at 100 C, 23 sec dwell time, Derivatization: Ambient, 100 l, 4 s dwell time

Conditions Column

Norm.
450 nm

3.0 2.5 2.0 1.5 1.0 0.5 0 250 300

330 nm

Excitation

Emission

350

400

450

500 Wavelength [nm]

Figure 14 Excitation (Ex=450 nm) and emission spectra (Em=330 nm) of the OPA-derivative of a carbamate

33

Environmental applications

Environmental applications

From the collected fluorescence spectra the lowest limit of detection is achievable when the detector is adjusted to Ex 230 nm/Em 450 nm or Ex 330 nm/Em 450 nm (figure 14). The individual carbamate residues do not shift the excitation and emission maxima significantly. The latter choice is slightly different from the literature where Ex 330 nm/ Em 465 nm is proposed. (ref. 5) Figure 15 compares the two wavelength settings found with the Agilent 1100 Series FLD. Watching the ratio of the two signals (Ex 230 nm/Em 450 nm and Ex 330 nm/Em 450 nm) allows control of peak purity.

Conditions See figure 14.

LU 40
4

35 30
1 6 2 3

1 2 3 4 5 6

Aldicarb sulfonate Aldicarb sulfoxide Oxamyl Methomyl 3-OH-Carbofuran Aldicarb 9 10

7 8 9 10 11 12

Propoxur Carbofuran 1-Naphthol Carbaryl Methiocarb BDMC

25 20 15 10

7 5

11

12

2.5 ng 230/450 nm

330/450 nm

5 Figure 15 Analysis of carbamates at two different excitation wavelengths, 230 and 330 nm 0 5 10 15 20 25 30 35 40 45 Time [min]

In a post-column reaction, the reagent is constantly flowing through the detector. To measure selectively the fluorescence of the derivatized carbamates, it is important to characterize the fluorescent properties of the reagent, impurities and the derivatized compounds completely. The fluorescence of the OPA-reagent in the mobile phase water/methanol is visible in a three-dimensional plot from a fluorescence scan (shown in figure 16). Excitation at 330 nm and emission at 450 nm are the best choices to achieve the

34

lowest limits of detection when monitoring selectively derivatized compounds independent of the background from the reagent.
Figure 16 Characterization of the mobile phase, including OPA reagent for carbamates The wavelength pair (Ex 330 nm/Em 450 nm) for quantitation of carbamates is separated from the reagents fluorescence. Chromatographic conditions were established and the fluorescence scan was taken under stop-flow conditions.

390

330

220 280

450

480

Food samples are complex matrices that need some sample preparation steps. In contrast, water can be clean enough to be injected directly. With high injection volumes, detection limits down to 100 ppt (S/N > 2) can be achieved (figure 17).
Conditions See figure 14.
1 2 3 4 5 5 4 Aldicarb sulfonate Oxamyl Methomyl 3-OH-Carbofuran Aldicarb 6 7 8 9 900 l 6 7 8 9 10 Propoxur Carbofuran 1-Naphthol Carbaryl Methiocarb

LU 19
1 3 2

18 17

10

16 15
800 l

Figure 17 Analysis of carbamate standard (250 ppt) in water With an injection volume of 800 to 900 l, a detection limit of 100 ppt can be achieved (S/N > 2).

330/450 nm

14 10 15 20 25 30 35 Time [min]

35

Environmental applications

Environmental applications

Conclusions
Optimized wavelength settings allow superior limits of detection. Changes in chromatographic conditions may change optimum wavelengths. Simultaneous multi wavelength detection enables chromatographers to control peak purity by watching the ratio of the two signals. Information on reagent properties and possible fluorescent impurities ensures reliable quantitative results.

36

Glyphosate

One of the most often used non-selective and post-emergence herbicides today is glyphosate (commonly found under the retail names of Roundup and Basta). Because of an increasing number of plants that are genetically engineered to resist glyphosate, the quantities applied may increase. Monitoring of glyphosate in soil, food and water is therefore becoming mandatory. The method of choice for analyzing glyphosate and its metabolite aminomethyl phosphonic acid (AMPA) is postcolumn derivatization based on a two-step mechanismoxidation with hypochlorit and reaction with o-phthalaldehyde. (ref.6) Several standard methods are currently being optimized, including DIN 38 407 F22 in Germany.

Experiments and results

Similar to the carbamate analysis, the optimum wavelengths for derivatizing glyphosate and AMPA are 230 nm excitation and 450 nm emission, as shown in figure 18. Because of the very high fluorescence background from the hypochlorit solution and OPA, the excitation wavelength 340 nm results in a signal-to noise ratio two times higher than the excitation wavelength 230 nm (see figure 19), although Ex 230 nm gives a much higher intensity. The limit of detection can be lowered to 5 pg (S/N>2). Depending on the injection volume, this is equivalent to 0.5 g/l (500 ppt) or even lower (see figure 20).

37

Environmental applications

Environmental applications

Conditions for figures 18, 19 and 20 Column Pickering Glyphosate column, 150 4.6 nm, Mobile phase A, 5 mM KH2PO4, pH 2.0, B, 100 % 5 mM potassium hydroxide Gradient 015 min, 100 % A, 1517 min, 100 % B, 1725 min, 100 % A, Flow rate 0.4 ml/min Column temperature 55 C FLD settings PMT 12, response time 4 s, step size 5 nm, Post-column conditions in the Pickering system: 0.3 ml/min Pickerings hypochlorite reagent for oxidation, 0.3 ml/min of Pickerings OPA reagent for derivatization, reactor for oxidation at 36 C, 500 l, 43 s dwell time Derivatization: Ambient, 100 l, 4 s dwell time

Norm. 200 175 150 125 100 75 50 25 0 200

230 nm

450 nm

Excitation

340 nm

Emission

250

300

350

400

450 Wavelength [nm]

Figure 18 Fluorescence spectra of a glyphosate derivative 10 ng/ml glyphosate dissolved in water).

Norm. 1000 800 600 400 200

Figure 19 Analysis of glyphosate and AMPA at different wavelengths Excitation at 340 nm offers a lower noise compared to Ex 230 nm.

340/450 nm 230/450 nm

2.5

7.5

10

12.5

15

17.5 Time [min]

38

LU 44.2 44.1 44.0 43.9 43.8 43.7 43.6

Figure 20 Trace level analysis of glyphosate and AMPA Injection of 500 ppt glyphosate and AMPA with a10-l injection volume (5 pg absolute)

Glyphosate AMPA

43.5 0 2.5 5 7.5 10 12.5 15

340/450 nm

17.5 20 Time [min]

Conclusions
Excitation of glyphosate can be achieved in the UV or visible range. Excitation at the 340 nm offers better selectivity because excitation at 230 nm gives high background noise and results in higher limits of detection. Glyphosate and AMPA can be detected down to the ppt level.

39

Environmental applications

Food applications

Mycotoxins

Aflatoxins and ochratoxin A belong to a large family of compounds produced by funghi. These mycotoxins are highly toxic compounds to protect themselves against other organisms. All kinds of plant tissue can be growth media for funghi, and therefore all types of food can be contaminated with mycotoxins. Storage conditions define the extent of fungal growth. Aflatoxins are known to cause degradation of fruits and vegetables. Ochratoxine is the prominent mycotoxin found in cereals, flour and figs. Because of the carcinogenic, teratogenic and mutagenic character of mycotoxins, food samples require careful control down to trace levels. A suitable clean-up procedure and optimized fluorescence or mass spectrometric detection are fundamental in achieving the required limits of detection in the low parts per billion (ppb) range.

Aflatoxins B1/B2 and G1/G2

Experiments and results

Two FLD runs produce optimum excitation and emission wavelengths, as described in chapter 2. The fluorescence spectra shown in figure 21 illustrate the result. Both the B- and G-type aflatoxins show similar spectra. The optimum excitation wavelength for both is 365 nm. The optimum emission wavelengths are different, 455 nm for the G-type and 445 nm for the B-type. These wavelengths deviate from the literature (ref. 7), which may be due to differences in experimental conditions such as the pH, the eluent composition or the instrumentation used. With the optimized conditions listed below, the limit of detection is down in the low nanogram range, as shown in figure 22. This means that a sample of only a few grams can be sufficient to detect at the low ppb level.

40

Figure 21 Fluorescence spectra for aflatoxins G2 and B2 Reference standards were dissolved in methanol (step size 5 nm).

Norm. 9 8 6 4

Aflatoxin B2

365 nm

445 nm

Excitation

Emission

2 0 200 Norm. 5 4 3 2 1 0 -1 200 250 300 350 400 450 500 Time [min]
Excitation Emission

250

300

350

400

450

500 Wavelength [nm]

Aflatoxin G2

365 nm

455 nm

Conditions Column

LU 12 10 8 6 4
0.3 ng G2

Hypersil ODS 100*2.1 mm, 3 m Mobile phase H2O/MeOH/CH3CN = 63/26/11 Flow rate 0.3 ml/min Column temperature 25 C Injection volume 5 l FLD settings PMT 12, response time 4 s, step size 5 nm, Figure 22 Analysis of aflatoxins at two different wavelengths

0.3 ng B2

1 ng G1

2 0

365/445 nm 365/460 nm

1 ng B1

10

12

14

16 18 Time [min]

41

Food applications

Food applications

Ochratoxin A

Ochratoxin A is found in degrading plant materials as a product of aspergillus or penicillium funghi. Through the food chain, this compound may become enriched in animal tissue and act as a cancerogenic. It can also be a substance that is directly toxic.

Experiments and results

The chromatogram in figure 23 shows a 125 pg of ochratoxine A at two different excitation wavelengths, 230 nm and 333 nm, with lower limits of detection at 230 nm but better selectivity over the matrix background at 333 nm.
Conditions Column Ochratoxin A

Zorbax SB C18, 150 2 mm, 3.5 m Mobile phase Water/acetonitrile = 50/50 Flow rate 0.4 ml/min Column temperature 40 C Injection volume 5 l FLD settings PMT 12, response time 4 s, step size 5 nm

LU 1.2 1.1 1.0 0.9 0.8 0.7 0.6

230/460 nm

Figure 23 Simultaneous multi wavelength detection for ochratoxine analysis 125 pg ochrotoxine was injected. The most appropriate wavelength can be chosen for quantitation depending on matrix conditions.

333/460 nm

0.5 0 2 4 6 8 10 12 14 Time [min]

Conclusions
Optimization of wavelength settings is mandatory if any change in experimental setup or chromatographic conditions occurs. The ability to detect simultaneously at multiple wavelengths helps to obtain the best limits of detection and selectivity for the different species of aflatoxins. Ochratoxine A may be excited in the UV or visible range depending on the need for a higher signal or a more stable baseline.

42

Vitamins B2 and B6

Vitamins are essential to avoid human and animal malnutrition. Improper storage may rapidly damage the natural vitamin content of food products or supplemental pharmaceutical formulations. Vitamins are classified as either water-soluble or fat-soluble compounds. Each is analyzed by different methods. Among the water-soluble vitamins, only B2 (riboflavin and phosphorylated riboflavins) and B6 (pyridoxamine, pyridoxal and, pyridoxine) show fluorescence. All compounds are separated in a single run. With previous fluorescence detectors they were detected using time-programmed wavelength switching. (ref.8)

Experiments and results

The optimal wavelengths for B6 vitamins are Ex 270/Em 400 nm and for vitamin B2 Ex 270/Em 530nm, as shown in figure 24. The detection limit for B2 is down to 20 pg; the detection limit for B6 is 200 pg (S/N > 2).
Figure 24 Excitation and emission spectra of vitamins B2 and B6 Spectra were extracted from two FLD runs for excitation and emission spectra (1 g/ml dissolved in water, step size 5 nm).

Norm. 35 30 25 20 15 10 5 0 200 Norm. 35 30 25 20 15 10 5 0 200

262 nm 362 nm

450 nm

530 nm

Excitation

Emission

250

300
280 nm

350

400
400 nm

450

500 550 Wavelength [nm]

Excitation

Emission

250

300

350

400

450

550 500 Wavelength [nm]

43

Food applications

Food applications

During metabolism as well as during sample preparation from complex matrices, vitamins are subject to various transformations. The goal is therefore to monitor precursors and modified species as well as the main compounds in a single run. Figure 25 demonstrates this for vitamins B2 and B6. The small impurities close to Riboflavine (seen in figure 26) can be identified through excitation spectra as belonging to the vitamin B2 complex. Reference standards help to identify them as monophosphates and diphosphates.

Zorbax SB 50 2.0 mm, 5 m Mobile phase A = 0.005 mM KH2PO4, pH 2.5 - H2SO4, B = acetonitrile Gradient 025 % B in 10 min Flow rate 0.5 ml/min Column temperature 35 C Injection volume 5 l FLD settings Response time 4 s Figure 25 Analysis of vitamins B2 and B6 Multi wavelength detection is used to detect both vitamins selectively and sensitive.

Conditions Column

LU Pryridoxamine 80 Pryridoxal Pryridoxine 60

Riboflavine

40

20

270/400 nm 270/530 nm 0 2 4 6 8 10 Time [min]

44

Conditions See figure 25.

LU 40 30 20 10

1 2 3 4 5 6

Pyridoxamine Pyridoxal Pyridoxine Riboflavine-diphosphate Riboflavine-5'monophosphate Riboflavine 5

Norm. 80 70

60 50 40 30 20 10 0 250 300 350 400 450 Wavelength [nm]

Figure 26 Confirmation of byproducts of vitamin B2 FLD excitation spectra (the red line is 5monophosphat, the blue line is riboflavin) show the similarity of spectra from the phosphorylated and unphosphorylated riboflavin (20 g/ml dissolved in mobile phase A).

0 1 -10 0

23

270/530 nm

10

12

14 Time[min]

Conclusions
The fluorescent vitamins B2 and B6 can be analyzed selectively at the pg level. Because of the difference in excitation and emission maxima, simultaneous multi wavelength detection is essential to detect both vitamins and byproducts at trace levels. Online spectra ensure confirmation of minor compounds throughout the complete HPLC run.

45

Food applications

Pharmaceutical applications

Quinidine

Quinidine occurs as two stereoisomers and has been used in anti-malaria and antiarrhythmic drugs since the beginning of the twentieth century. Before that, American Indians used plant material containing quinidine for its antipyretic activity. This compound has a complex chemical structure containing a fluorophor and groups (tert. amine) that can be protonated (illustrated in figure 27). An HPLC separation with well shaped peaks requires a buffered eluent as described in the literature. (ref. 9) With a new generation of more stable column materials (such as Zorbax Stablebond), this compound has to be analyzed under acidic conditions. As much as retention times change with the type of stationary phase, a different pH of the mobile phase changes UV absorption and fluorescence behavior.

N HO H CH3O

N
Figure 27 Chemical structure of quinidine The arrows indicate the functional groups that can be protonated.

Experiments and results

As pH decreases, quinidine shows a bathochrome shift: The emission wavelength changes from 380 nm to 450 nm as the pH shifts from pH 7 to pH 2.5 (shown in figure 28). This is important in maintaining optimum limits of detection. If slight changes in pH occur, these can contribute to non-reproducible separation and quantitation results. Recording spectra can help to view these changes. Consequently, spectral data can be used in routine work to check separation conditionsa prerequisite for reproducing quantitative results.

46

Conditions Method 1 pH 7 Column Purosphere 125 4 mm, 5 m Mobile phase H2O pH 7 (H2SO4)/ acetonitrile Flow rate 0.6 ml/min Column temperature 40 C Injection volume 1 l FLD settings PMT 12, response time 4 s, step size 5 nm Method 2 pH 2.5 Column BDS 100 2.1 mm, 3 m Mobile phase H2O pH 2.5 (H2SO4)/ acetonitrile Flow rate 0.3 ml/min Column temperature 40 C Injection volume 1 l FLD settings PMT 12, response time 4 s, step size 5 nm

Norm. 35 30 25 20 15 10 5

380 nm

450 nm

pH 7

pH 2.5

Emission

0 350 400 450 500

Emission

550 600 Wavelength [nm]

Figure 28 Influence of pH on emission spectra for quinidine

Conclusions
Fluorescence spectra are an excellent indicator of pH changes in the eluent. Quantitation of acidic or basic type substances requires strict control of pH not only to control retention times but for reliable quantitation as well.

47

Pharmaceutical applications

Pharmaceutical applications

Warfarin

Warfarin is an anticoagulant drug that is used in post-surgery treatment. The chemical structure derives from coumarine and has phenolic character. In the literature, warfarin is analyzed mainly at neutral conditions (phosphate buffer pH 7.5) with fluorescence measured at Ex 290 nm/Em 390 nm or Ex 310 nm/Em 370 nm. (ref.10)

Experiments and results

Warfarin can be analyzed on a Zorbax Stablebond column at pH 2.5. Under these conditions, excitation and emission maxima shifted to 272 nm and 355 nm, respectively (shown in figure 29). The resulting comparison between the analysis based on literature and actual optimized fluorescence wavelengths is shown in figure 30. Actual optimized fluorescence wavelengths show about three times better limits of detection. Compared to UV detection, the Agilent 1100 Series FLD has a limit of detection about 20 times lower.

Conditions Column

Norm. 272 nm 1.75 1.50 1.25 1.00 Excitation 0.75 0.50 0.25 0 200 250 300 350 400 450 500 Wavelength [nm] Emission 355 nm

Zorbax SB C18, 50 2.1 mm, 3 m Mobile phase 0.005 M KH2PO4, pH 3/acetonitrile Gradient 20 % B to 80 % B in 10 min Wash 80 % B to 20 % B in 2 min Column temperature 25 C Injection volume 1 l FLD settings PMT 12, response time 4 s, step size 5 nm

Figure 29 Fluorescence spectra for warfarin under acidic conditions (pH=3) 10 g/ml dissolved in phosphate buffer.

48

Conditions See figure 29.

LU 3.5 3.0 2.5 2.0 1.5

10 ng Warfarin

272/355 nm

290/390 nm

1.0
Figure 30 Analysis of warfarin Response under literature conditions (ref. 10) and actual optimized fluorescence wavelengths.
accord. Lit.

0.5 0 1 2 3 4 5 6 7 8 9 Time [min]

Conclusions
New types of columns that produce different retention behavior require a rework of chromatographic conditions. This may induce significant shifts in fluorescence spectra, which in turn can influence the limits of detection with the fluorescence detector. In routine analysis, fluorescence spectra can be taken automatically and reviewed to check for accidental changes in chromatographic conditions.

49

Pharmaceutical applications

Pharmaceutical applications

Amino Acids

Amino acids are essential components for biological systems and have been found already during early stages of life on earth. For human nutrition a set of essential amino acids is needed that cannot be formed in the body but must be taken up from the daily food. As building blocks of the proteins they form the vast majority of all enzymes in biochemical transformations. A wide range of decease and drug research is targeted on protein biochemistry to understand the role of amino acids, peptides and proteins. After acidic or enzymatic hydrolysis amino acid composition can be determined using reversed phase columns which will give typically a better resolution than ion chromatography. For sensitive detection amino acids are derivatized in a two-step pre-column derivatization. A post-column derivatisation is suitable for higher selectivity as required for complex biological samples. Fluorescence detection is used for concentrations below 100 pmol/l. UV-detection is a choice for higher concentrations up to the nanogram range. A standard HPLC system can be used for this straightforward and cost-effective approach to amino acid analysis.

Compound (pmol) Asp Glu Ser His Gly Thr Ala Arg Tyr Cys-SS-Cys Val Met Phe Ile Leu Lys Pro

LOD for FLD (fmol) 19 18 21 29 21 21 20 17 19 17 16 17 16 18 57 22

FLD

FLD

0.139 0.155 0.156 0.155 0.118 0.113 0.120 0.094 0.062 not measured 0.058 0.045 0.048 0.050 0.040 0.060 0.044

0.924 0.576 1.015 1.778 1.124 0.739 0.767 0.905 1.614 0.919 1.236 1.079 0.759 0.952 5.107 4.379

Experimental and results

Pre-column derivatisation of primary amino acids is achieved with ortho-phthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC) is used for secondary amino acids. Seventeen different amino acids that are all found in protein hydrolysates were analyzed as shown in figure 31. A wavelength switching program is used to detect Proline. The limits of detection for the amino acids are listed in table 4 based on a signal-to-noise > 2. This is close to 100 times more sensitive than UV-detection at 338nm. Retention time precision is typically below 0.2 % and peak area precision is typically below 5 %.

Table 4 LODs for fluorescence and UV detection

50

Arg

200 2.1 mm AA column and guard column Mobile phase A = 20 mMol NaAc + 0.018% TEA adjusted to pH 7.2 with 1-2 % acetic acid, B = 20 % of 100 mMol NaAc adjusted to pH 7.2 with 1-2 % acetic acid + 40 % ACN and 40 % MeOH Flow rate 0.45 ml/min Gradient start with: 100 %A, at 17 min 60 %B, at 18 min, 100 %B, at 18.1 min flow 0.45, at 18.5 min flow 0.8, at 23.9 min flow 0.8, at 24 min 100%B and flow 0.45, at 25 min 0%B Oven temp. 40 C Post time 5 min Injector program 1 Draw 5.0 l from vial 10borate buffer 2 Draw 1.0 l from vial 11OPA reagent 3 Draw 0.0 l from vial 12water 4 Draw 1.0 l from sample 5 Draw 0.0 l from vial 12water 6 Mix 8 l in air, max speed, six times 7 Draw 1.0 l from vial 14FMOC 8 Draw 0.0 l from vial 12water 9 Mix 9 l in air, max speed, 3 times 10 Inject FLD settings Excitation 340 nm Emission 450 nm PTM gain 12 at 14.5 min Excitation =266 nm Emission = 305 nm PTM Gain 11

LU 175 150 125 100 75 50 25 0 0

Asp

Ala

Try

Val Met Phe Ile Leu

Glu

Conditions Column

10 pmol standard

10

12

14

16

18 Time [min]

Figure 31 Analysis of 10 pmol/l amino acids with fluorescence detection

Conclusion
Reversed phase chromatography combined with precolumn derivatization is an ideal tool for automated costeffective amino acid analysis on a standard HPLC setup. With the use of OPA and FMOC and fluorescence detection it is possible to push the limits of detection signifantly below 100 femtomoles for most amino acids. This technique is worked out to offer precise results for protein hydrolysates in a routine laboratory.

51

Pharmaceutical applications

Ser

His

Gly Thr

Cys-SS-Cys

Lys

Pro

References

1. Lakowicz, J.R., Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983. 2. Schuster, R., A comparison of pre- and post-column derivatization for the analysis of glyphosate, Agilent Technologies Application Note 5091-3621E, 1991. 3. Froehlich, P., Internatl Laboratory, No. 10, 42-44,1989. 4. Brownrigg, J.T. and Sullivan, M. J., Spectroscopy, Vol.1, No.2, 1989. 5. Pickering Laboratories, Product brochure, Publ. No. B-CA 4, 1992. 6. Pickering Laboratories, Application Note, Publ. No. B-CA 5, 1993. 7. Official Methods of Analysis, Food Compositions; Additives, Natural Contaminants, 15th edition: AOAC: Arlington, VA, Vol. 2.; AOAC Official method 980.20, aflatoxins in cotton seed products, 1990. 8. Mc Calley, D.V., J. Chrom., 357, 221, 1986. 9. Chu, Y-Q. and Wainer, I.W., Pharm. Res. 5, 680, 1988. 10. Gratzfeld-Huesgen, A. and Schuster, R., HPLC for Food Analysis, Agilent Technologies Application Primer, 5968-9345E, 2000. 11. Gratzfeld-Huesgen, A., Sensitive and Reliable Amino Acids Analysis in Protein Hydrolysates using the Agilent 1100 Series HPLC, Agilent Technologies Technical Note 5968-5658E, 1999.

52

A
aflatoxins, 4041 B1/B2, 4041 G1/G2 4041 amino acids, 50 AMPA, 3739 antioxidant, 31 anthracene, 14, 22, 26 applications, 29 ASTM, 14 2-amino-3-hydroxyphenazine (AHP), 23,24

G
german standard method DIN, 30, 37 glyphosate, 3739 grating, 1112

Q
qualitative analysis, 24 quantitative analysis, 2526 quinidine, 18, 19, 4647

I
impurities, 17, 23, 25 isofluorescence plot, 13, 17, 22

R
Raman, 14,17 Raleigh, 17 riboflavine, 4344

L
lamp, 11 Xenon, 1112 light sources, 23 limit of detection (LOD), 12, 1618, 23, 25, 30, 31, 33, 34, 35, 36, 37, 39, 40, 42, 43, 46, 48,

S
selectivity, 7, 18, 23, 25 specifications, 14 spectral libraries, 13 spectral mode, 67, 32 spectral range, 21 stray light, 1112 Raman, 11, 17 Raleigh, 17

B
bandwidth, 1011 2-benzimidazole carbamic acid methylester (MBC), 23, 24

C
carbamates, 3336 cut-off filter, 11, 17 cuvette, 12, 17, 19, 20 chromophore, 10

M
mass spectrometric, 6, 26, 40 match factor, 27 method development, 1627 2-benzimidazole carbamic acid methylester (MBC), 23, 24 mobile phase, 17, 34, 35, 46 monochromator, 1112 multi wavelength detection, 16, 25, 3132, 36, 42, 4445, mycotoxins, 40

T
three-dimensional plot, 12, 13, 17, 21, 34 trichlorophenol, 31, 32

D
data handling, 1213 data analysis, 13 derivatization, 33 detector diode array, 6, 13, 23, 26, 31 mass spectrometric, 6, 26, 40 2,3-diamino-phenazine (DAP), 23, 24 DIN method, 30, 37

U
UV/Visible absorption spectra, 6

V
vitamins, 4345 B2/B6, 4345

O
o-phthalaldehyde (OPA), 3335 ochratoxin A, 42

W
warfarin, 48 wavelength, 12

E
EPA methods, 30, 33 emission monochromator, 1112 spectra, 6, 1014, 1627, 33, 43, 46, 47 wavelength, 1014, 1625, 40 excitation monochromator, 1112 spectra, 6, 1014, 1626, 33, 43 wavelength, 1014, 1625, 40,

P
peak, 2527 purity, 13, 27, 34 identity, 26 pesticides, 33 photo-multiplier, 11 photodiode, 11 Pickering, 38 polynuclear aromatic hydrocarbons (PNA), 21, 25, 3032 precolumn derivatization, postcolumn derivatization, 33, 37 purity factor, 27 pyridoxal, 43, 44 pyridoxamine, 43, 44 pyridoxine, 43, 44

X
Xenon flash lamp, 1112

Z
Zorbax, 46, 48

F
fluorescence spectra, 13, 19, 25, 34, 38, 40, 41, 48, 49 fluorescence scan, 12, 17, 21, 34 fluorophore, 21, 33, 46

53

Index

www.agilent.com/chem
Copyright 2000 Agilent Technologies The information in this publication is subject to change without notice. All Rights Reserved. Reproduction, adaptation or translation without prior written permission is prohibited, except as allowed under the copyright laws. Printed in Germany 01/00 Publication Number 5968-9346E