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HPLC–NMR, Pharmaceutical Applications☆
HPLC–NMR, Pharmaceutical Applications JP Shockcor, Du Pont Pharmaceuticals Co, Newark, NJ, USA ã 2017 Elsevier Ltd. All rights reserved.
Q T T1 T2 Vc Vs g t
Symbols b B0 f I n n N
receiver bandwidth magnetic field strength coil filling factor spin quantum number oversampling factor preamplifier noise figure number of detected nuclei
Abbreviations ADC COSY HMBC HMQC HPLC MS
analog-to-digital converter correlation spectroscopy heteronuclear multiple bond correlation heteronuclear multiple bond correlation high-performance liquid chromatography mass spectroscopy
Introduction High-performance liquid chromatography (HPLC) has become a routine tool for the separation of complex mixtures. However, structural information on substances separated using HPLC is limited by the detector system employed. The most common HPLC detectors, refractive index, UV (ultraviolet), radiochemical, fluorescence, and electrochemical, provide little or no structural information. As a result, structure elucidation required the isolation of the analyte from the matrix, followed by off-line spectroscopic characterization. With the advent of HPLC-MS (mass spectrometry), the ability to detect and identify substances at low concentrations without the need for an isolation step became possible. Although this has simplified structure elucidation to a great extent, there are often circumstances where HPLC-MS alone is insufficient for complete characterization of a compound and further studies by nuclear magnetic resonance (NMR) are required. Logically, the next step in instrument development would be directly coupling HPLC and NMR yielding the hyphenated technique HPLC-NMR.
History In the late 1970s and early 1980s, a number of attempts to couple these techniques were carried out. However, these This article is reproduced from the previous edition, Copyright 1999, Elsevier Ltd.
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
coil quality factor temperature spin–lattice relaxation time spin–spin relaxation time detector-coil volume sample volume gyromagnetic ratio flow rate
NMR RF S/N tNMRc TOCSY UV WET
nuclear magnetic resonance radio frequency signal-to-noise total NMR chromatogram total correlation spectroscopy ultraviolet water suppression enhanced through T1 effects
studies suffered from the low sensitivity of the NMR spectrometer systems then available. Also, because of dynamic-range problems, there was a need to use expensive deuterated solvents for the HPLC because the solvent suppression methods in use at that time could not cope with fully protonated solvents. The reduction in HPLC-NMR to routine use was slow in developing and not practically achieved until technical improvements in electronics, higher magnetic fields strengths, advanced solvent suppression sequences, and improved instrumental sensitivity made it feasible to interface an HPLC directly to an NMR spectrometer. In addition, investigations into coupling other types of chromatography with NMR have been carried out. These include capillary electrophoresis, capillary electrochromatography, supercritical fluid chromatography, and capillary HPLC. However, there are no commercially available systems based on these separations.
Technical Improvements There have been many major technical improvements in NMR sensitivity in recent years. Foremost has been the development of ultra-high-field-strength magnets. High-dynamic range analog-to-digital converters (ADCs), which give benefits in situations where large solvent peaks are present along with small analyte signals, and digital filtering techniques that allow spectral windows to be limited to include only the region of interest, without the problem of signals and noise from outside this spectral region folding in, have also contributed to
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increases in sensitivity. Another important improvement is oversampling. That is, the collection of digital data points at a rate faster than that required to satisfy the Nyquist criterion of twice the highest desired spectral frequency. In theory, for an oversampling factor of n, a gain in dynamic range of log2(n) is obtained, that is, for an oversampling of eight times, an effective gain of 3 bits in ADC resolution of the signals results. In practice, the oversampled signal is simply averaged over the n measurements to restore the same number of data points corresponding to the Nyquist criterion. This prevents folding of noise or artifacts that would have been in the extended spectral region, resulting from a consequence of the oversampling being equivalent to a spectral region n times wider than required to satisfy the Nyquist criterion. Thus, a second consequence of oversampling is an improved signal-to-noise (S/N) ratio from removal of folded noise when the spectral region is truncated.
The simplest method of operation is continuous-flow detection. This mode of operation is generally only feasible when using 1H or 19F NMR for detection unless enriched compounds are used. If on-flow detection is required during a solvent gradient elution, the NMR resonance positions of the solvent peaks will shift as the solvent proportions change. For effective solvent suppression, it is therefore necessary to determine these solvent resonance frequencies as the chromatographic run proceeds. This is accomplished by measuring a single exploratory scan as soon as a chromatographic peak is detected in real time during the chromatographic run and then applying solvent suppression irradiation at these frequencies as the peak elutes. The data from an on-flow experiment performed on a sample of human urine after dosing with paracetamol [1] are shown in Figure 2. The data are plotted in a pseudo-2-D format with the axes being the chemical shift in part per million and the retention time of the chromatographic run. Slices extracted from the 2-D plot show the 1-D spectra of the glucuronide [2] and sulfate [3] conjugates of paracetamol (Figure 3). O
The HPLC-NMR System A block diagram of a typical instrumental setup for HPLCNMR is shown in Figure 1. This comprises a high-resolution NMR spectrometer with its superconducting magnet into which is placed a dedicated NMR flow probe, a standard HPLC system controlled by PC-resident software, and a flow control unit that enables the system to be operated in four main modes as listed in the following text.
• • • •
HN
On-flow Stop-flow Time-sliced stop-flow Peak collection into capillary loops for postchromatographic analysis.
CH3
OH [1] Paracetamol
NMR spectrometer console
COM 3
Magnet 500 MHz
COM 1 COM 2 Analog signal
HPLC Injector
LC−NMR interface Loops
Column 2 10
Detector Optional mass spectrometer Figure 1 Block diagram of a typical HPLC-NMR system.
Fraction collector Direct line Waste
HPLC–NMR, Pharmaceutical Applications
If the retention times of the compounds to be separated are known, or if they can be detected using refractive index, UV (including diode arrays), radiochemical, or fluorescence detectors, stop-flow HPLC-NMR becomes an option. Upon detection the PC controlling the liquid chromatograph allows the pumps to continue running, moving the peak of the interest into the NMR probe. Once the pumps have stopped, normal high-resolution NMR is possible. It could be argued that the long length of capillary tubing connecting the HPLC system to the NMR probe will cause a significant loss of resolution in the separation. In fact this is not a problem. The NMR detection cell volume is typically 60–120 ml, and this represents the limiting factor in the chromatographic resolution. The practicality of the stop-flow approach has been amply demonstrated, and although several separate stops are often made in each chromatographic run, the quality of the resulting NMR spectra is such that good structural information can be obtained. Even long 2-D experiments, which provide correlation between NMR resonances, based on mutual spin–spin coupling such as correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and heteronuclear correlation studies such as heteronuclear multiple quantum coherence (HMQC) spectroscopy can be performed. It is in this stop-flow mode that HPLCNMR is most commonly used. There are two further special categories of stop-flow experiment. These comprise the ability to store eluting fractions in capillary loops for later off-line NMR study (‘peak picking’) and the very useful ability to stop the flow at short intervals over a chromatographic peak to ‘time slice’ different parts of a chromatographic run. This is analogous to the use of diodearray UV spectroscopy to obtain spectra from various positions within an eluting peak to determine peak purity. The timeslicing methods may be useful if there is poor chromatographic separation, if the compounds under study have weak or no UV chromophores or if the exact chromatographic retention time is unknown. It is also possible to time slice through an entire
O
CH3
HN
H HOOC
H O
HO HO
O OH
H H
H
[2] Glucuronide conjugate of paracetamol
O
CH3
HN
OSO3H [3] Sulfate conjugate of paracetamol
Sulfate conjugate Glucuronide conjugate
8.5
7.5 1
6.5
143
5.5
4.5
3.5
2.5
1.5 ppm
Figure 2 Pseudo-2-D plot of on-flow H HPLC-NMR data obtained on human urine after dosing with paracetamol [1]. The resonances from the glucuronide [2] and the sulfate [3] conjugate metabolites of paracetamol are indicated.
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Sulfate conjugate
Glucuronide conjugate
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
ppm
Figure 3 One-dimensional slices extracted from the on-flow experiment shown in Figure 2. The top trace is the sulfate conjugate [2] and the bottom trace is the glucuronide conjugate [3].
chromatographic run producing the equivalent of an on-flow experiment with higher signal to noise. The data from such a time-slicing experiment have been referred to as a total NMR chromatogram (tNMRc). In all the modes described earlier, programmed gradient elution profiles can be performed and no compromise needs to be made in the chromatographic conditions, with the exception of those outlined in the following section.
HPLC-NMR Solvents and Chromatographic Considerations Another major problem in development of HPLC-NMR involved the use of deuterated solvents. Conventional highresolution NMR spectroscopy routinely uses deuterated solvents. However, these solvents are thought to be prohibitively expensive if they have to be used for elution in HPLC-NMR because of the volumes involved. It is always possible to use solvents such as CCl4, which contain no protons, for normalphase chromatography, but this then requires the use of HPLCNMR probes that have a separate external deuterium lock channel. Since most chromatographic methods are reverse phase, they involve the use of H2O and an organic solvent such as acetonitrile or methanol. These give rise to large signals in the NMR spectrum that can obscure the spectrum of the analyte. The solution to this problem is solvent suppression that can be performed quite efficiently on modern NMR spectrometers. So efficiently, in fact, that the use of deuterated solvents is no longer a necessity. However, D2O is used rather than H2O to make the multiple solvent suppression easier and to serve as the field-lock substance. The cost of D2O is relatively modest.
One of the most common organic solvents used in reversephase HPLC separations is acetonitrile, which gives rise to a singlet resonance in the 1H NMR spectrum at about d 2.0. This singlet and that arising from residual H2O in D2O can be easily suppressed by presaturation. However, these suppression methods based on presaturation leave the 13C satellite peaks from the 1.1% of molecules with the naturally abundant 13C isotope at the methyl carbon. These satellite peaks are often much larger than the analyte peaks of interest in an HPLCNMR study and so it is often necessary to suppress these also. This has been achieved in two ways. It is possible to set the suppression irradiation frequency over the central peak and the two satellite peaks in a cyclic fashion or, if an inverse-geometry probe is used, which includes a 13C coil, then 13C decoupling is possible. This will collapse the satellite peaks under the central peak, enabling conventional single-frequency suppression. The Water suppression Enhanced through T1 effects (WET) solvent suppression method incorporates selective radio frequency (RF) pulses, pulsed-field gradients and optional 13C decoupling to provide a robust solvent suppression method well suited to HPLC-NMR. Figure 4 shows an example of WET solvent suppression on the 1H HPLC-NMR spectrum of the desmethyl–glucuronide conjugate of naproxen [4] from human urine. The top trace shows the unsuppressed spectrum, whereas the bottom trace shows the same sample with WET solvent suppression. Use of a fully deuterated solvent system, acetonitrile-d3 and D2O, is also an option when looking at very small quantities of material with resonances in the region near d 2.0. Although the cost might seem to be prohibitive, at approximately $1500 per liter for acetonitrile-d3, the solvents for a typical HPLC run using both D2O and acetonitrile-d3 cost only $12–15.
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7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm
Figure 4 An example of solvent suppression using WET. The top trace shows the data with no solvent suppression and the bottom trace shows the data with WET solvent suppression applied.
Other chromatographic considerations, outlined in the following text, should be considered when designing an HPLC separation method.
•
Use buffers that have as few 1H NMR resonances as possible. – Trifluoroacetic acid. – Ammonium phosphate. – Ammonium acetate. – Deuterated formic acid ($130 per 5 g).
•
Use ionpair reagents that have as few 1H NMR resonances as possible. – Ionpairs with n-butyl groups create four additional resonances. – Ionpairs with t-butyl groups create one additional resonance.
•
Whenever possible use HPLC-gradient methods. – Run at a 5% change per minute maximum. – Use columns that will be suitable for the earlier criteria. – Reverse-phase Cn. – 2.0–4.6 mm diameter.
25 C. The total probe volume including the inlet line and the outlet to waste was measured to be 800 ml. Samples were injected directly into the NMR probe and hence the tests were of probe sensitivity rather than the overall sensitivity of HPLCNMR. The S/N ratio is defined as the peak height of a given signal multiplied by 2.5 and divided by the peak-to-peak height of the noise measured over a given spectral range of 200 Hz. Spectra were measured on a standard sample of 0.1% ethylbenzene in CDCl3. The 1H S/N calculation, based on the signal for the methylene quartet at 2.7 ppm, gave a value of 237:1. A typical organic molecule 30 -deoxy-30 -azidothymidine (AZT) [5] was used to test the absolute detection limit of the same HPLC-NMR probe. Having determined the total volume of the NMR probe system to be 800 ml, a solution containing 1 mg of AZT in 120 ml was injected directly into the probe. The solvent was 80% D2O–20% acetonitrile. The S/N ratio was measured on the ribose H10 proton in the 1H NMR spectrum at d 6.2. The detection limit for this 1H signal was defined as a signal height to peak-to-peak noise ratio of 3.0 (i.e., a S/N of 7.5 by the definition given earlier). Thus, a detection limit of 500 ng in 64 scans was calculated. H3C
Sensitivity of HPLC-NMR The sensitivity of HPLC-NMR and the expected detection limits have been estimated for both 1D 1H NMR and 2-D NMR. All NMR experiments were carried out using a Bruker Avance 500 NMR spectrometer operating at a 1H observation frequency of 500.13 MHz. The spectral data were measured using a dedicated 1H–13C inverse-geometry HPLC-NMR probe, with a 120 ml active volume. All NMR spectra were measured at
O
OH Glucuronide−O [4] Desmethyl−glucuronide conjugate of naproxen
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HPLC–NMR, Pharmaceutical Applications
•
O CH3
•
HN
O
•
S=N ¼ g:NIðI þ 1Þ:ðB0 =T Þ3=2 :f :ðQV s =bÞ1=2 :n1
N
HO
[1]
Therefore, S/N can be increased by doing the following: O
• • • N3 [5] AZT
AZT [5] was also used to test the 1H sensitivity limit for a H–1H 2D TOCSY spectrum. A spectrum was obtained using a sample of 1 mg of AZT in 120 ml of 80% D2O–20% acetonitrile and acquired over 4.0 h. Examination of peak volumes shows that the detection limit of this experiment for 16 h of acquisition (i.e., overnight) is approximately 500 ng. The data acquisition parameters used were designed to ensure full T1 relaxation so that any relative integral values would be quantitative. However, these are not the parameters, which give maximum S/N values. If the 1H NMR relaxation times are assumed to be approximately 1.5 s, and if the data had been collected in a manner designed to yield maximum S/ N (i.e., with a pulse repetition rate of approximately 1.3 T1), then this would result in a S/N enhancement by a factor of approximately 1.4. In addition, because of the faster pulsing rate, more scans could be accumulated in the same experimental time, leading to a further S/N enhancement factor of approximately 1.7. Thus, the overall S/N might be expected to increase by a factor of approximately 2.4. In addition, for the 2-D experiments, it is possible to halve the number of T1 increments and extrapolate the data using linear prediction. This allows the acquisition of twice as many scans per increment for the same overall experiment time, resulting in a 1.4 increase in S/N. These detection limits will be lowered as new technical advances in NMR become available. These include higher magnetic field strengths and the future availability of HPLC-NMR cyroprobes, probes cooled to cryogenic temperatures, which reduce the noise figure and can provide as much as a 4.0 increase in S/N. 1
HPLC-NMR Probe Design The flow probe is the heart of the HPLC-NMR system. The main design criteria for such probes have been defined as follows:
•
NMR line broadening from magnetic susceptibility and other cell-wall effects should be avoided. The coil design and position should provide the highest possible NMR sensitivity. The achievable S/N ratio of a NMR detector is a function of a number of parameters expressed in eqn [1].
The geometry of the NMR cell should allow flow characteristics that give spectral resolution sufficient to allow spincoupled multiplets to be resolved. This enables detailed structural elucidation to be carried out.
• • • •
Increase the sample volume and hence the number of detected nuclei, N. Increase the magnetic field strength B0. Increase the filling factor, f, of the NMR coil (this is Vs/Vc, where Vs is the sample volume and Vc is the volume inside the detector coil). Increase the quality factor, Q, of the RF coil. Reduce the receiver bandwidth b. Operate at reduced temperature T. Improve preamplifiers by reducing their noise figure n.
All these are for a given nucleus with gyromagnetic ratio g and spin quantum number I. For HPLC-NMR probes, there is a compromise that has to be made in that the detection volume should be as small as possible to get optimum chromatographic resolution and hence this can only be compensated for by increasing the filling factor. This is achieved by fixing the RF coil directly onto the outside of the NMR cell. If this is done, it is of course not possible to spin the sample to improve magnetic field inhomogeneities. However, in practice this is not a problem because the smaller the sample volume, the better the shimming. Additionally, the use of a computer-controlled orthogonal shim sets has reduced the necessity to spin the sample by greatly improving nonspinning line shapes. One major factor in determining the sensitivity or peak heights is the observed line shape. If the peaks have wide bases, then a significant part of the signal intensity is found in this part of the peak and poor S/N results. Thus, good shimming, especially with respect to the ‘hump test’ (the linewidth of the chloroform 1H NMR peak at the height of the 13C satellite signals relative to that at 20% of the height of the 13C satellite signals) is a prerequisite for good S/N and essential to successful HPLC-NMR operation. In superconducting magnets, the main magnetic field is parallel to the long axis of the NMR flow cell, and the flow direction and the RF are applied to a Helmholtz coil on the side of the probe insert. This coil design has a lower Q than a horizontal solenoid coil. A horizontal coil would be preferred on sensitivity grounds, but shim systems are designed for vertical sample tubes, as normally samples are inserted vertically from the top of the magnet. The NMR flow cell fixed in a vertical position has the advantage of inducing laminar flow that helps remove air bubbles from the cell. NMR flow cells have so far had much larger volumes than, for example, UV detection cells because of the lower sensitivity of NMR. The effects that large-volume NMR cells have on chromatographic peak dispersion have been investigated using a modified fluorescence detector. The selection of the detection volume of an NMR flow cell is a compromise between the needs of the NMR sensitivity and the HPLC
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HPLC–NMR, Pharmaceutical Applications resolution. Typically, the optimum detection volume is 1–5 ml when an HPLC column of 2504 mm is used. In contrast, the usual detected volume in a conventional NMR tube is rarely less than 60 ml. It has been shown that the NMR resonances are broadened at different flow rates using detectors with a range of volumes. At flow rates of 1 ml min1, an increase of 0.14 Hz in linewidth is obtained only with flow cell volumes <120 ml. The broadening is due to an effective shortening of the spin–spin relaxation time, T2, due to flow. 1=T2 ðobsÞ ¼ 1=T2 þ 1=t
Applications HPLC-NMR has been applied to a wide range of analytical problems. These include
• • • • •
Cl
O
[2]
Thus, where t is the flow rate. The selection of an HPLCNMR probe is thus a compromise between sensitivity and resolution. The two most common flow cell volumes are 60 and 120 ml, respectively. In general, the 60 ml flow cells are best for on-flow experiments and stop-flow using small-diameter columns with low flow rates. The 120 ml flow cell is more suited to stop-flow experiments using conventional HPLC columns and flow rates.
•
F3C
The characterization of endogenous and xenobiotic metabolites directly from a biological matrix. Characterization of metabolites from in vitro studies. The dynamic study of reactive metabolites. Characterization of natural products from complex mixtures. Polymer characterization. Characterization of reaction impurities from small- and large-scale systems.
To illustrate the utility of HPLC-NMR and provide examples of typical data from HPLC-NMR experiments, the characterization of the human urinary metabolites of the anti-HIV drug efavirenz (Sustiva) is described in the following text. Efavirenz [6] is a nonnucleoside inhibitor of HIV-1 reverse transcriptase. The metabolism of efavirenz in rats was studied using directly coupled HPLC-NMR-MS. This hyphenated technique utilizes an eluent splitter to also incorporate a mass spectrometer into the HPLC-NMR system to obtain additional information on molecular weight and to act as a detector for selection of peaks on which to perform NMR experiments. The sample for analysis was prepared by solid-phase extraction of 5 ml of urine obtained from rats after dosing with 800 mg kg1 of efavirenz, with 0–24 h collection. The extract was dried and reconstituted with 100 ml of 80% D2O and 20% acetonitrile-d3. A 40 ml injection was made onto a 3.9150 Waters Symmetry C18 column. A gradient elution from 80% D2O and 20% acetonitrile-d3 to 50% D2O and 50% acetonitrile-d3 over 20 min at a flow rate of 0.8 ml min1 was employed for separation. Using a splitter immediately after the column, 95% of the sample went to the UV detector and onto the NMR spectrometer, whereas 5% went to a Finnigan LC ion-trap mass spectrometer equipped, with an ESI probe operating in the positive-ion mode. The system was plumbed to allow the peak to reach the UV detector and the mass spectrometer at the same time.
5
7 O
N H
8
[6] Efavirenz
The initial experiment, an on-flow 19F detected HPLCNMR-MS run, takes advantage of the CF3 group present in the parent drug. Since there are no endogenous fluorinated compounds in rat urine, responses in the spectrum must arise from metabolites of efavirenz. This experiment provided the retention times of the metabolites that were of sufficient concentration to be detected. The 19F chemical shift and the mass of the metabolites were also obtained from this single experiment. Figure 5 shows the data as a pseudo-2-D plot. The peaks of interest are labeled with their molecular mass. The next phase of the experimental procedure is to obtain stop-flow 1H HPLC-NMR spectra at the retention times determined in the on-flow 19F HPLC-NMR experiment. The 1-D and TOCSY spectra obtained on the minor metabolite with retention time 14.2 min, mass 605 Da, and 19F chemical shift 84.6 ppm are shown in Figure 6. The mass-spectral data showed a loss of 80 Da from the parent mass indicative of SO3 loss. The loss of the methine proton from the cyclopropyl ring, seen clearly from the TOCSY spectrum, and the downfield shift of the remaining methylene protons indicate addition of OH to the cyclopropyl ring. The singlet at d 6.47 is consistent with reduction in the alkyne. The spin system observed between d 3 and d 4 ppm is consistent with cysteinylglycine conjugation. On the basis of these data and the observed molecular mass of 605 Da, this peak is assigned as [7]. O 4' HO
O HN S 3' S
1' F3C
2'
5
CI
O
OH
7 8 HSO3O
N H [7]
O
NH2
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HPLC–NMR, Pharmaceutical Applications
Time (min) 28 26 24
MW 507
MW 491
22 20 18 16
MW 605
14 12 −82.5
−82.0
−83.0
−83.5
−84.0
−84.5 ppm
Figure 5 Pseudo-2-D plot of on-flow 19H HPLC-NMR-MS data obtained on rat urine after dosing with efavirenz. The data supply the 19F chemical shift, retention time, and mass of the metabolites.
3',4'
1' 7
2'
5 ppm
1 2 Cysteinylglycine 3 4 5 6 7 8 8
7
6
5
8
3
2
1
ppm
Figure 6 One-dimensional and TOCSY HPLC-NMR spectra of the 8-O-sulfate, cysteinylglycine diconjugate of efavirenz.
HPLC–NMR, Pharmaceutical Applications
5
1'
7
5'
149
2' , 3' , 4'
ppm Cyclopropyl 1 2 3 Glucuronide 4 5 6 7 8 8
7
6
5
8
3
2
1
ppm
Figure 7 One-dimensional and TOCSY HPLC-NMR spectra of the 8-O-glucuronide conjugate of efavirenz.
The 1-D and TOCSY data obtained on the major metabolite with retention time 20.5 min, mass 507 Da, and 19F chemical shift 83.3 ppm are shown in Figure 7. These data clearly show the presence of a glucuronide conjugate. The observed changes in the aromatic region of the spectrum are consistent with the 8-O-glucuronide conjugate of efavirenz [8].
several resonances to a glucuronide conjugate. The assignment of this metabolite as the N-glucuronide conjugate [9] is consistent with the chemical shift of H10 , d 4.75, and with the observed molecular mass.
5
Cl
F3C O
F3C 5
CI
7
O 7
N H
8
O HO
1' HO
4' O
3'
OH 2′
5′ 4′
OH
3′
OH
OH O [9] N-glucuronide conjugate of efavirenz
2'
5'
O 1
O
O O
N 8
OH
OH
[8] 8-O-glucuronide conjugate of efavirenz
Summary Coeluting with the major metabolite is a component with mass 491 Da and 19F chemical shift 82.1 ppm. Using the mass spectrometer as a detector, the stop-flow was executed when the desired mass, 491 Da, was observed. Although the spectrum (Figure 8) is dominated by the major metabolite, the 8-O-glucuronide conjugate of efavirenz, it is possible to assign
The experiments described serve to illustrate how HPLC-NMR can rapidly provide information on the structure of drug metabolites. These same methods have been employed in the study of endogenous compounds in biofluids, natural products, polymers, and many other complex mixtures.
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5,7,8
7.5
1'
7.0
6.5
6.0
5.5
5.0
5'
4.5
4.0
2',3',4'
3.5
3.0
2.5
2.0
1.5
1.0
ppm
Figure 8 The H HPLC-NMR spectrum of the N-glucuronide conjugate of efavirenz shown as the minor component, along with the major component the 8-O-glucuronide conjugate. 1
The high cost of the HPLC-NMR system is of course a factor that must be taken into consideration. Since most laboratories have seen the value of NMR in its traditional form, it is only necessary to add an HPLC system, an appropriate flow probe, and a flow control unit to an existing spectrometer to enable HPLC-NMR experiments to be performed. The cost of these accessories, or even the cost of a complete system dedicated to HPLC-NMR, is offset by the efficiency of the method. The laborious extraction of minor components from complex mixtures followed by off-line analysis or in the case of synthetic drugs, the synthesis of radiochemically labeled materials with the numerous problems associated with handling and disposing of radiolabeled samples make the cost associated with HPLC-NMR less of a factor. There are a number of limitations to the utility of HPLCNMR. The greatest of these is the limitation on the amount of material that can be loaded on the column and consequently moved into the NMR probe. HPLC-NMR is not well suited to the less-sensitive NMR techniques like, which provides critical structural information via long-range interactions. Thus, it is still necessary to isolate minor components from complex mixtures on occasion, and HPLC-NMR should not be viewed as a replacement for conventional NMR. It is a tool that enhances the utility of NMR and like any tool it can be used properly and effectively or misused. Although it has experimental limitations when compared to conventional NMR, many structural problems can be solved using HPLC-NMR without resorting to tedious isolation methods.
The advantages of HPLC-NMR can be summarized as follows:
• • • •
Speed of analysis by elimination of the isolation step. Ability to obtain structural information and high-quality spectral data from complex matrices. The lack of need for perfect chromatographic separation. Synergy when directly coupled with MS.
The direct coupling of chromatography, principally HPLC, to NMR spectroscopy continues to be an area of active research and application. Many papers have resulted from a wide range of applications, mainly pharmaceutical studies of degradation and metabolism, and natural product characterization. With the advent of cryogenically cooled probes and higher sensitivity NMR instruments, the field continues to be very active. An up-to-date summary of the current instrumental methodologies applicable can be found in other articles in this encyclopedia via the ‘See also’ list.
See also: Atomic Spectroscopy, Biomedical Applications; Atomic Spectroscopy, Pharmaceutical Applications; Biofluids Studied by NMR Spectroscopy; Biological Applications of Hyperpolarized 13C NMR; Circular Dichroism and ORD, Biomacromolecular Applications; Counterfeit Drugs Studied by NMR; Drug Metabolism Studied Using NMR Spectroscopy; Enantiomeric Purity Studied Using NMR; Fragment-Based Drug Design by NMR; Hyphenated NMR, Methods and Applications; IR, Biological Applications; IR, Medical Science
HPLC–NMR, Pharmaceutical Applications
Applications; Mass Spectrometry in Drug Metabolism: Principles and Common Practice; Medical Applications of Mass Spectrometry; MRI Applications, Biological; NMR Applications, Solution state 19F; NMR Spectrometers; NMR Spectroscopy in the Evaluation of Drug Safety; Raman Optical Activity, Macromolecule and Biological Molecule Applications; Raman Spectroscopy, Medical Applications: A New Look Inside Human Body With Raman Imaging; Solvent Suppression Methods in NMR Spectroscopy; Spectroscopic Methods in Drug Quality Control and Development; Spectroscopy for Process Analytical Technology (PAT); Spectroscopy in Biotechnology Research and Development; Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals; UV-Visible Absorption Spectroscopy, Biomacromolecular Applications; Vibrational Spectroscopy Applications in Drugs Analysis.
Further Reading Albert K (1995) On-line use of NMR detection in separation chemistry. Journal of Chromatography A 703: 123–147. Albert K (1995) Direct on-line coupling of capillary electrophoresis and 1H NMR spectroscopy. Angewandte Chemie International Edition in English 34: 641–642. Albert K, Braumann U, Tseng L-H, et al. (1994) On-line coupling of supercritical fluid chromatography and proton high-field nuclear magnetic resonance spectroscopy. Analytical Chemistry 66: 3042–3046. Albert K, Kunst M, Bayer E, Spraul M, and Bermel W (1989) Reverse-phase highperformance liquid chromatography-nuclear magnetic resonance on-line coupling with solvent non-excitation. Journal of Chromatography 463: 355. Behnke B, Schlotterbeck G, Tallarek U, et al. (1996) Capillary HPLC-NMR coupling: High resolution NMR spectroscopy in the nanoliter scale. Analytical Chemistry 68: 1110–1115. Griffiths L (1995) Optimization of NMR and HPLC conditions for LC-NMR. Analytical Chemistry 67: 4091–4095.
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Laude DA Jr and Wilkins CL (1984) Direct-linked analytical scale high-performance liquid chromatography/nuclear magnetic resonance spectroscopy. Analytical Chemistry 56: 2471–2475. Lindon JC, Farrant RD, Sanderson PN, et al. (1995) Separation and characterization of components of peptide libraries using on-flow coupled HPLC-NMR spectroscopy. Magnetic Resonance in Chemistry 33: 857–863. Lindon JC, Nicholson JK, and Wilson ID (1996) Direct coupling of chromatographic separations to NMR spectroscopy. Progress in Nuclear Magnetic Resonance Spectroscopy 29: 1–49. Lindon JC, Nicholson JK, Sidelmann UG, and Wilson ID (1997) Directly coupled HPLC-NMR and its application to drug metabolism. Drug Metabolism Reviews 29: 705–746. Pullen FS, Swanson AG, Newman MJ, and Richards DS (1995) “On-line” liquid chromatography/nuclear magnetic resonance mass spectrometry—A powerful spectroscopic tool for the analysis of mixtures of pharmaceutical interest. Rapid Communications in Mass Spectrometry 9: 1003–1006. Shockcor JP, Unger SE, Wilson ID, Foxall PJD, Nicholson JK, and Lindon JC (1996) Combined HPLC, NMR spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine. Analytical Chemistry 68: 4431–4435. Shockcor JP, Wurm RM, Frick LW, et al. (1996) HPLC-NMR identification of the human urinary metabolites of (–)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine, a nucleoside analogue active against human immunodeficiency virus (HIV). Xenobiotica 26: 189–199. Smallcombe SH, Patt SL, and Keifer PA (1995) WET solvent suppression and its application to LC-NMR and high resolution NMR spectroscopy. Journal of Magnetic Resonance Series A 117: 295–303. Sweatman BC, Farrant RD, Sanderson PN, et al. (1995) Evaluation of the detection limits of directly coupled 600 MHz 1H and 1H-13C HPLC-NMR spectroscopy. Journal of Magnetic Resonance Analysis 1: 9–12. Wolfender J-L, Rodriguez S, and Hostettmann K (1998) Liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance spectroscopy for the screening of plant constituents. Journal of Chromatography A 794: 299–316.
Q T T1 T2 Vc Vs g t
Symbols b B0 f I n n N
receiver bandwidth magnetic field strength coil filling factor spin quantum number oversampling factor preamplifier noise figure number of detected nuclei
Abbreviations ADC COSY HMBC HMQC HPLC MS
analog-to-digital converter correlation spectroscopy heteronuclear multiple bond correlation heteronuclear multiple bond correlation high-performance liquid chromatography mass spectroscopy
Introduction High-performance liquid chromatography (HPLC) has become a routine tool for the separation of complex mixtures. However, structural information on substances separated using HPLC is limited by the detector system employed. The most common HPLC detectors, refractive index, UV (ultraviolet), radiochemical, fluorescence, and electrochemical, provide little or no structural information. As a result, structure elucidation required the isolation of the analyte from the matrix, followed by off-line spectroscopic characterization. With the advent of HPLC-MS (mass spectrometry), the ability to detect and identify substances at low concentrations without the need for an isolation step became possible. Although this has simplified structure elucidation to a great extent, there are often circumstances where HPLC-MS alone is insufficient for complete characterization of a compound and further studies by nuclear magnetic resonance (NMR) are required. Logically, the next step in instrument development would be directly coupling HPLC and NMR yielding the hyphenated technique HPLC-NMR.
History In the late 1970s and early 1980s, a number of attempts to couple these techniques were carried out. However, these This article is reproduced from the previous edition, Copyright 1999, Elsevier Ltd.
Encyclopedia of Spectroscopy and Spectrometry, Third Edition
coil quality factor temperature spin–lattice relaxation time spin–spin relaxation time detector-coil volume sample volume gyromagnetic ratio flow rate
NMR RF S/N tNMRc TOCSY UV WET
nuclear magnetic resonance radio frequency signal-to-noise total NMR chromatogram total correlation spectroscopy ultraviolet water suppression enhanced through T1 effects
studies suffered from the low sensitivity of the NMR spectrometer systems then available. Also, because of dynamic-range problems, there was a need to use expensive deuterated solvents for the HPLC because the solvent suppression methods in use at that time could not cope with fully protonated solvents. The reduction in HPLC-NMR to routine use was slow in developing and not practically achieved until technical improvements in electronics, higher magnetic fields strengths, advanced solvent suppression sequences, and improved instrumental sensitivity made it feasible to interface an HPLC directly to an NMR spectrometer. In addition, investigations into coupling other types of chromatography with NMR have been carried out. These include capillary electrophoresis, capillary electrochromatography, supercritical fluid chromatography, and capillary HPLC. However, there are no commercially available systems based on these separations.
Technical Improvements There have been many major technical improvements in NMR sensitivity in recent years. Foremost has been the development of ultra-high-field-strength magnets. High-dynamic range analog-to-digital converters (ADCs), which give benefits in situations where large solvent peaks are present along with small analyte signals, and digital filtering techniques that allow spectral windows to be limited to include only the region of interest, without the problem of signals and noise from outside this spectral region folding in, have also contributed to
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increases in sensitivity. Another important improvement is oversampling. That is, the collection of digital data points at a rate faster than that required to satisfy the Nyquist criterion of twice the highest desired spectral frequency. In theory, for an oversampling factor of n, a gain in dynamic range of log2(n) is obtained, that is, for an oversampling of eight times, an effective gain of 3 bits in ADC resolution of the signals results. In practice, the oversampled signal is simply averaged over the n measurements to restore the same number of data points corresponding to the Nyquist criterion. This prevents folding of noise or artifacts that would have been in the extended spectral region, resulting from a consequence of the oversampling being equivalent to a spectral region n times wider than required to satisfy the Nyquist criterion. Thus, a second consequence of oversampling is an improved signal-to-noise (S/N) ratio from removal of folded noise when the spectral region is truncated.
The simplest method of operation is continuous-flow detection. This mode of operation is generally only feasible when using 1H or 19F NMR for detection unless enriched compounds are used. If on-flow detection is required during a solvent gradient elution, the NMR resonance positions of the solvent peaks will shift as the solvent proportions change. For effective solvent suppression, it is therefore necessary to determine these solvent resonance frequencies as the chromatographic run proceeds. This is accomplished by measuring a single exploratory scan as soon as a chromatographic peak is detected in real time during the chromatographic run and then applying solvent suppression irradiation at these frequencies as the peak elutes. The data from an on-flow experiment performed on a sample of human urine after dosing with paracetamol [1] are shown in Figure 2. The data are plotted in a pseudo-2-D format with the axes being the chemical shift in part per million and the retention time of the chromatographic run. Slices extracted from the 2-D plot show the 1-D spectra of the glucuronide [2] and sulfate [3] conjugates of paracetamol (Figure 3). O
The HPLC-NMR System A block diagram of a typical instrumental setup for HPLCNMR is shown in Figure 1. This comprises a high-resolution NMR spectrometer with its superconducting magnet into which is placed a dedicated NMR flow probe, a standard HPLC system controlled by PC-resident software, and a flow control unit that enables the system to be operated in four main modes as listed in the following text.
• • • •
HN
On-flow Stop-flow Time-sliced stop-flow Peak collection into capillary loops for postchromatographic analysis.
CH3
OH [1] Paracetamol
NMR spectrometer console
COM 3
Magnet 500 MHz
COM 1 COM 2 Analog signal
HPLC Injector
LC−NMR interface Loops
Column 2 10
Detector Optional mass spectrometer Figure 1 Block diagram of a typical HPLC-NMR system.
Fraction collector Direct line Waste
HPLC–NMR, Pharmaceutical Applications
If the retention times of the compounds to be separated are known, or if they can be detected using refractive index, UV (including diode arrays), radiochemical, or fluorescence detectors, stop-flow HPLC-NMR becomes an option. Upon detection the PC controlling the liquid chromatograph allows the pumps to continue running, moving the peak of the interest into the NMR probe. Once the pumps have stopped, normal high-resolution NMR is possible. It could be argued that the long length of capillary tubing connecting the HPLC system to the NMR probe will cause a significant loss of resolution in the separation. In fact this is not a problem. The NMR detection cell volume is typically 60–120 ml, and this represents the limiting factor in the chromatographic resolution. The practicality of the stop-flow approach has been amply demonstrated, and although several separate stops are often made in each chromatographic run, the quality of the resulting NMR spectra is such that good structural information can be obtained. Even long 2-D experiments, which provide correlation between NMR resonances, based on mutual spin–spin coupling such as correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and heteronuclear correlation studies such as heteronuclear multiple quantum coherence (HMQC) spectroscopy can be performed. It is in this stop-flow mode that HPLCNMR is most commonly used. There are two further special categories of stop-flow experiment. These comprise the ability to store eluting fractions in capillary loops for later off-line NMR study (‘peak picking’) and the very useful ability to stop the flow at short intervals over a chromatographic peak to ‘time slice’ different parts of a chromatographic run. This is analogous to the use of diodearray UV spectroscopy to obtain spectra from various positions within an eluting peak to determine peak purity. The timeslicing methods may be useful if there is poor chromatographic separation, if the compounds under study have weak or no UV chromophores or if the exact chromatographic retention time is unknown. It is also possible to time slice through an entire
O
CH3
HN
H HOOC
H O
HO HO
O OH
H H
H
[2] Glucuronide conjugate of paracetamol
O
CH3
HN
OSO3H [3] Sulfate conjugate of paracetamol
Sulfate conjugate Glucuronide conjugate
8.5
7.5 1
6.5
143
5.5
4.5
3.5
2.5
1.5 ppm
Figure 2 Pseudo-2-D plot of on-flow H HPLC-NMR data obtained on human urine after dosing with paracetamol [1]. The resonances from the glucuronide [2] and the sulfate [3] conjugate metabolites of paracetamol are indicated.
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Sulfate conjugate
Glucuronide conjugate
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
ppm
Figure 3 One-dimensional slices extracted from the on-flow experiment shown in Figure 2. The top trace is the sulfate conjugate [2] and the bottom trace is the glucuronide conjugate [3].
chromatographic run producing the equivalent of an on-flow experiment with higher signal to noise. The data from such a time-slicing experiment have been referred to as a total NMR chromatogram (tNMRc). In all the modes described earlier, programmed gradient elution profiles can be performed and no compromise needs to be made in the chromatographic conditions, with the exception of those outlined in the following section.
HPLC-NMR Solvents and Chromatographic Considerations Another major problem in development of HPLC-NMR involved the use of deuterated solvents. Conventional highresolution NMR spectroscopy routinely uses deuterated solvents. However, these solvents are thought to be prohibitively expensive if they have to be used for elution in HPLC-NMR because of the volumes involved. It is always possible to use solvents such as CCl4, which contain no protons, for normalphase chromatography, but this then requires the use of HPLCNMR probes that have a separate external deuterium lock channel. Since most chromatographic methods are reverse phase, they involve the use of H2O and an organic solvent such as acetonitrile or methanol. These give rise to large signals in the NMR spectrum that can obscure the spectrum of the analyte. The solution to this problem is solvent suppression that can be performed quite efficiently on modern NMR spectrometers. So efficiently, in fact, that the use of deuterated solvents is no longer a necessity. However, D2O is used rather than H2O to make the multiple solvent suppression easier and to serve as the field-lock substance. The cost of D2O is relatively modest.
One of the most common organic solvents used in reversephase HPLC separations is acetonitrile, which gives rise to a singlet resonance in the 1H NMR spectrum at about d 2.0. This singlet and that arising from residual H2O in D2O can be easily suppressed by presaturation. However, these suppression methods based on presaturation leave the 13C satellite peaks from the 1.1% of molecules with the naturally abundant 13C isotope at the methyl carbon. These satellite peaks are often much larger than the analyte peaks of interest in an HPLCNMR study and so it is often necessary to suppress these also. This has been achieved in two ways. It is possible to set the suppression irradiation frequency over the central peak and the two satellite peaks in a cyclic fashion or, if an inverse-geometry probe is used, which includes a 13C coil, then 13C decoupling is possible. This will collapse the satellite peaks under the central peak, enabling conventional single-frequency suppression. The Water suppression Enhanced through T1 effects (WET) solvent suppression method incorporates selective radio frequency (RF) pulses, pulsed-field gradients and optional 13C decoupling to provide a robust solvent suppression method well suited to HPLC-NMR. Figure 4 shows an example of WET solvent suppression on the 1H HPLC-NMR spectrum of the desmethyl–glucuronide conjugate of naproxen [4] from human urine. The top trace shows the unsuppressed spectrum, whereas the bottom trace shows the same sample with WET solvent suppression. Use of a fully deuterated solvent system, acetonitrile-d3 and D2O, is also an option when looking at very small quantities of material with resonances in the region near d 2.0. Although the cost might seem to be prohibitive, at approximately $1500 per liter for acetonitrile-d3, the solvents for a typical HPLC run using both D2O and acetonitrile-d3 cost only $12–15.
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7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm
Figure 4 An example of solvent suppression using WET. The top trace shows the data with no solvent suppression and the bottom trace shows the data with WET solvent suppression applied.
Other chromatographic considerations, outlined in the following text, should be considered when designing an HPLC separation method.
•
Use buffers that have as few 1H NMR resonances as possible. – Trifluoroacetic acid. – Ammonium phosphate. – Ammonium acetate. – Deuterated formic acid ($130 per 5 g).
•
Use ionpair reagents that have as few 1H NMR resonances as possible. – Ionpairs with n-butyl groups create four additional resonances. – Ionpairs with t-butyl groups create one additional resonance.
•
Whenever possible use HPLC-gradient methods. – Run at a 5% change per minute maximum. – Use columns that will be suitable for the earlier criteria. – Reverse-phase Cn. – 2.0–4.6 mm diameter.
25 C. The total probe volume including the inlet line and the outlet to waste was measured to be 800 ml. Samples were injected directly into the NMR probe and hence the tests were of probe sensitivity rather than the overall sensitivity of HPLCNMR. The S/N ratio is defined as the peak height of a given signal multiplied by 2.5 and divided by the peak-to-peak height of the noise measured over a given spectral range of 200 Hz. Spectra were measured on a standard sample of 0.1% ethylbenzene in CDCl3. The 1H S/N calculation, based on the signal for the methylene quartet at 2.7 ppm, gave a value of 237:1. A typical organic molecule 30 -deoxy-30 -azidothymidine (AZT) [5] was used to test the absolute detection limit of the same HPLC-NMR probe. Having determined the total volume of the NMR probe system to be 800 ml, a solution containing 1 mg of AZT in 120 ml was injected directly into the probe. The solvent was 80% D2O–20% acetonitrile. The S/N ratio was measured on the ribose H10 proton in the 1H NMR spectrum at d 6.2. The detection limit for this 1H signal was defined as a signal height to peak-to-peak noise ratio of 3.0 (i.e., a S/N of 7.5 by the definition given earlier). Thus, a detection limit of 500 ng in 64 scans was calculated. H3C
Sensitivity of HPLC-NMR The sensitivity of HPLC-NMR and the expected detection limits have been estimated for both 1D 1H NMR and 2-D NMR. All NMR experiments were carried out using a Bruker Avance 500 NMR spectrometer operating at a 1H observation frequency of 500.13 MHz. The spectral data were measured using a dedicated 1H–13C inverse-geometry HPLC-NMR probe, with a 120 ml active volume. All NMR spectra were measured at
O
OH Glucuronide−O [4] Desmethyl−glucuronide conjugate of naproxen
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HPLC–NMR, Pharmaceutical Applications
•
O CH3
•
HN
O
•
S=N ¼ g:NIðI þ 1Þ:ðB0 =T Þ3=2 :f :ðQV s =bÞ1=2 :n1
N
HO
[1]
Therefore, S/N can be increased by doing the following: O
• • • N3 [5] AZT
AZT [5] was also used to test the 1H sensitivity limit for a H–1H 2D TOCSY spectrum. A spectrum was obtained using a sample of 1 mg of AZT in 120 ml of 80% D2O–20% acetonitrile and acquired over 4.0 h. Examination of peak volumes shows that the detection limit of this experiment for 16 h of acquisition (i.e., overnight) is approximately 500 ng. The data acquisition parameters used were designed to ensure full T1 relaxation so that any relative integral values would be quantitative. However, these are not the parameters, which give maximum S/N values. If the 1H NMR relaxation times are assumed to be approximately 1.5 s, and if the data had been collected in a manner designed to yield maximum S/ N (i.e., with a pulse repetition rate of approximately 1.3 T1), then this would result in a S/N enhancement by a factor of approximately 1.4. In addition, because of the faster pulsing rate, more scans could be accumulated in the same experimental time, leading to a further S/N enhancement factor of approximately 1.7. Thus, the overall S/N might be expected to increase by a factor of approximately 2.4. In addition, for the 2-D experiments, it is possible to halve the number of T1 increments and extrapolate the data using linear prediction. This allows the acquisition of twice as many scans per increment for the same overall experiment time, resulting in a 1.4 increase in S/N. These detection limits will be lowered as new technical advances in NMR become available. These include higher magnetic field strengths and the future availability of HPLC-NMR cyroprobes, probes cooled to cryogenic temperatures, which reduce the noise figure and can provide as much as a 4.0 increase in S/N. 1
HPLC-NMR Probe Design The flow probe is the heart of the HPLC-NMR system. The main design criteria for such probes have been defined as follows:
•
NMR line broadening from magnetic susceptibility and other cell-wall effects should be avoided. The coil design and position should provide the highest possible NMR sensitivity. The achievable S/N ratio of a NMR detector is a function of a number of parameters expressed in eqn [1].
The geometry of the NMR cell should allow flow characteristics that give spectral resolution sufficient to allow spincoupled multiplets to be resolved. This enables detailed structural elucidation to be carried out.
• • • •
Increase the sample volume and hence the number of detected nuclei, N. Increase the magnetic field strength B0. Increase the filling factor, f, of the NMR coil (this is Vs/Vc, where Vs is the sample volume and Vc is the volume inside the detector coil). Increase the quality factor, Q, of the RF coil. Reduce the receiver bandwidth b. Operate at reduced temperature T. Improve preamplifiers by reducing their noise figure n.
All these are for a given nucleus with gyromagnetic ratio g and spin quantum number I. For HPLC-NMR probes, there is a compromise that has to be made in that the detection volume should be as small as possible to get optimum chromatographic resolution and hence this can only be compensated for by increasing the filling factor. This is achieved by fixing the RF coil directly onto the outside of the NMR cell. If this is done, it is of course not possible to spin the sample to improve magnetic field inhomogeneities. However, in practice this is not a problem because the smaller the sample volume, the better the shimming. Additionally, the use of a computer-controlled orthogonal shim sets has reduced the necessity to spin the sample by greatly improving nonspinning line shapes. One major factor in determining the sensitivity or peak heights is the observed line shape. If the peaks have wide bases, then a significant part of the signal intensity is found in this part of the peak and poor S/N results. Thus, good shimming, especially with respect to the ‘hump test’ (the linewidth of the chloroform 1H NMR peak at the height of the 13C satellite signals relative to that at 20% of the height of the 13C satellite signals) is a prerequisite for good S/N and essential to successful HPLC-NMR operation. In superconducting magnets, the main magnetic field is parallel to the long axis of the NMR flow cell, and the flow direction and the RF are applied to a Helmholtz coil on the side of the probe insert. This coil design has a lower Q than a horizontal solenoid coil. A horizontal coil would be preferred on sensitivity grounds, but shim systems are designed for vertical sample tubes, as normally samples are inserted vertically from the top of the magnet. The NMR flow cell fixed in a vertical position has the advantage of inducing laminar flow that helps remove air bubbles from the cell. NMR flow cells have so far had much larger volumes than, for example, UV detection cells because of the lower sensitivity of NMR. The effects that large-volume NMR cells have on chromatographic peak dispersion have been investigated using a modified fluorescence detector. The selection of the detection volume of an NMR flow cell is a compromise between the needs of the NMR sensitivity and the HPLC
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HPLC–NMR, Pharmaceutical Applications resolution. Typically, the optimum detection volume is 1–5 ml when an HPLC column of 2504 mm is used. In contrast, the usual detected volume in a conventional NMR tube is rarely less than 60 ml. It has been shown that the NMR resonances are broadened at different flow rates using detectors with a range of volumes. At flow rates of 1 ml min1, an increase of 0.14 Hz in linewidth is obtained only with flow cell volumes <120 ml. The broadening is due to an effective shortening of the spin–spin relaxation time, T2, due to flow. 1=T2 ðobsÞ ¼ 1=T2 þ 1=t
Applications HPLC-NMR has been applied to a wide range of analytical problems. These include
• • • • •
Cl
O
[2]
Thus, where t is the flow rate. The selection of an HPLCNMR probe is thus a compromise between sensitivity and resolution. The two most common flow cell volumes are 60 and 120 ml, respectively. In general, the 60 ml flow cells are best for on-flow experiments and stop-flow using small-diameter columns with low flow rates. The 120 ml flow cell is more suited to stop-flow experiments using conventional HPLC columns and flow rates.
•
F3C
The characterization of endogenous and xenobiotic metabolites directly from a biological matrix. Characterization of metabolites from in vitro studies. The dynamic study of reactive metabolites. Characterization of natural products from complex mixtures. Polymer characterization. Characterization of reaction impurities from small- and large-scale systems.
To illustrate the utility of HPLC-NMR and provide examples of typical data from HPLC-NMR experiments, the characterization of the human urinary metabolites of the anti-HIV drug efavirenz (Sustiva) is described in the following text. Efavirenz [6] is a nonnucleoside inhibitor of HIV-1 reverse transcriptase. The metabolism of efavirenz in rats was studied using directly coupled HPLC-NMR-MS. This hyphenated technique utilizes an eluent splitter to also incorporate a mass spectrometer into the HPLC-NMR system to obtain additional information on molecular weight and to act as a detector for selection of peaks on which to perform NMR experiments. The sample for analysis was prepared by solid-phase extraction of 5 ml of urine obtained from rats after dosing with 800 mg kg1 of efavirenz, with 0–24 h collection. The extract was dried and reconstituted with 100 ml of 80% D2O and 20% acetonitrile-d3. A 40 ml injection was made onto a 3.9150 Waters Symmetry C18 column. A gradient elution from 80% D2O and 20% acetonitrile-d3 to 50% D2O and 50% acetonitrile-d3 over 20 min at a flow rate of 0.8 ml min1 was employed for separation. Using a splitter immediately after the column, 95% of the sample went to the UV detector and onto the NMR spectrometer, whereas 5% went to a Finnigan LC ion-trap mass spectrometer equipped, with an ESI probe operating in the positive-ion mode. The system was plumbed to allow the peak to reach the UV detector and the mass spectrometer at the same time.
5
7 O
N H
8
[6] Efavirenz
The initial experiment, an on-flow 19F detected HPLCNMR-MS run, takes advantage of the CF3 group present in the parent drug. Since there are no endogenous fluorinated compounds in rat urine, responses in the spectrum must arise from metabolites of efavirenz. This experiment provided the retention times of the metabolites that were of sufficient concentration to be detected. The 19F chemical shift and the mass of the metabolites were also obtained from this single experiment. Figure 5 shows the data as a pseudo-2-D plot. The peaks of interest are labeled with their molecular mass. The next phase of the experimental procedure is to obtain stop-flow 1H HPLC-NMR spectra at the retention times determined in the on-flow 19F HPLC-NMR experiment. The 1-D and TOCSY spectra obtained on the minor metabolite with retention time 14.2 min, mass 605 Da, and 19F chemical shift 84.6 ppm are shown in Figure 6. The mass-spectral data showed a loss of 80 Da from the parent mass indicative of SO3 loss. The loss of the methine proton from the cyclopropyl ring, seen clearly from the TOCSY spectrum, and the downfield shift of the remaining methylene protons indicate addition of OH to the cyclopropyl ring. The singlet at d 6.47 is consistent with reduction in the alkyne. The spin system observed between d 3 and d 4 ppm is consistent with cysteinylglycine conjugation. On the basis of these data and the observed molecular mass of 605 Da, this peak is assigned as [7]. O 4' HO
O HN S 3' S
1' F3C
2'
5
CI
O
OH
7 8 HSO3O
N H [7]
O
NH2
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HPLC–NMR, Pharmaceutical Applications
Time (min) 28 26 24
MW 507
MW 491
22 20 18 16
MW 605
14 12 −82.5
−82.0
−83.0
−83.5
−84.0
−84.5 ppm
Figure 5 Pseudo-2-D plot of on-flow 19H HPLC-NMR-MS data obtained on rat urine after dosing with efavirenz. The data supply the 19F chemical shift, retention time, and mass of the metabolites.
3',4'
1' 7
2'
5 ppm
1 2 Cysteinylglycine 3 4 5 6 7 8 8
7
6
5
8
3
2
1
ppm
Figure 6 One-dimensional and TOCSY HPLC-NMR spectra of the 8-O-sulfate, cysteinylglycine diconjugate of efavirenz.
HPLC–NMR, Pharmaceutical Applications
5
1'
7
5'
149
2' , 3' , 4'
ppm Cyclopropyl 1 2 3 Glucuronide 4 5 6 7 8 8
7
6
5
8
3
2
1
ppm
Figure 7 One-dimensional and TOCSY HPLC-NMR spectra of the 8-O-glucuronide conjugate of efavirenz.
The 1-D and TOCSY data obtained on the major metabolite with retention time 20.5 min, mass 507 Da, and 19F chemical shift 83.3 ppm are shown in Figure 7. These data clearly show the presence of a glucuronide conjugate. The observed changes in the aromatic region of the spectrum are consistent with the 8-O-glucuronide conjugate of efavirenz [8].
several resonances to a glucuronide conjugate. The assignment of this metabolite as the N-glucuronide conjugate [9] is consistent with the chemical shift of H10 , d 4.75, and with the observed molecular mass.
5
Cl
F3C O
F3C 5
CI
7
O 7
N H
8
O HO
1' HO
4' O
3'
OH 2′
5′ 4′
OH
3′
OH
OH O [9] N-glucuronide conjugate of efavirenz
2'
5'
O 1
O
O O
N 8
OH
OH
[8] 8-O-glucuronide conjugate of efavirenz
Summary Coeluting with the major metabolite is a component with mass 491 Da and 19F chemical shift 82.1 ppm. Using the mass spectrometer as a detector, the stop-flow was executed when the desired mass, 491 Da, was observed. Although the spectrum (Figure 8) is dominated by the major metabolite, the 8-O-glucuronide conjugate of efavirenz, it is possible to assign
The experiments described serve to illustrate how HPLC-NMR can rapidly provide information on the structure of drug metabolites. These same methods have been employed in the study of endogenous compounds in biofluids, natural products, polymers, and many other complex mixtures.
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5,7,8
7.5
1'
7.0
6.5
6.0
5.5
5.0
5'
4.5
4.0
2',3',4'
3.5
3.0
2.5
2.0
1.5
1.0
ppm
Figure 8 The H HPLC-NMR spectrum of the N-glucuronide conjugate of efavirenz shown as the minor component, along with the major component the 8-O-glucuronide conjugate. 1
The high cost of the HPLC-NMR system is of course a factor that must be taken into consideration. Since most laboratories have seen the value of NMR in its traditional form, it is only necessary to add an HPLC system, an appropriate flow probe, and a flow control unit to an existing spectrometer to enable HPLC-NMR experiments to be performed. The cost of these accessories, or even the cost of a complete system dedicated to HPLC-NMR, is offset by the efficiency of the method. The laborious extraction of minor components from complex mixtures followed by off-line analysis or in the case of synthetic drugs, the synthesis of radiochemically labeled materials with the numerous problems associated with handling and disposing of radiolabeled samples make the cost associated with HPLC-NMR less of a factor. There are a number of limitations to the utility of HPLCNMR. The greatest of these is the limitation on the amount of material that can be loaded on the column and consequently moved into the NMR probe. HPLC-NMR is not well suited to the less-sensitive NMR techniques like, which provides critical structural information via long-range interactions. Thus, it is still necessary to isolate minor components from complex mixtures on occasion, and HPLC-NMR should not be viewed as a replacement for conventional NMR. It is a tool that enhances the utility of NMR and like any tool it can be used properly and effectively or misused. Although it has experimental limitations when compared to conventional NMR, many structural problems can be solved using HPLC-NMR without resorting to tedious isolation methods.
The advantages of HPLC-NMR can be summarized as follows:
• • • •
Speed of analysis by elimination of the isolation step. Ability to obtain structural information and high-quality spectral data from complex matrices. The lack of need for perfect chromatographic separation. Synergy when directly coupled with MS.
The direct coupling of chromatography, principally HPLC, to NMR spectroscopy continues to be an area of active research and application. Many papers have resulted from a wide range of applications, mainly pharmaceutical studies of degradation and metabolism, and natural product characterization. With the advent of cryogenically cooled probes and higher sensitivity NMR instruments, the field continues to be very active. An up-to-date summary of the current instrumental methodologies applicable can be found in other articles in this encyclopedia via the ‘See also’ list.
See also: Atomic Spectroscopy, Biomedical Applications; Atomic Spectroscopy, Pharmaceutical Applications; Biofluids Studied by NMR Spectroscopy; Biological Applications of Hyperpolarized 13C NMR; Circular Dichroism and ORD, Biomacromolecular Applications; Counterfeit Drugs Studied by NMR; Drug Metabolism Studied Using NMR Spectroscopy; Enantiomeric Purity Studied Using NMR; Fragment-Based Drug Design by NMR; Hyphenated NMR, Methods and Applications; IR, Biological Applications; IR, Medical Science
HPLC–NMR, Pharmaceutical Applications
Applications; Mass Spectrometry in Drug Metabolism: Principles and Common Practice; Medical Applications of Mass Spectrometry; MRI Applications, Biological; NMR Applications, Solution state 19F; NMR Spectrometers; NMR Spectroscopy in the Evaluation of Drug Safety; Raman Optical Activity, Macromolecule and Biological Molecule Applications; Raman Spectroscopy, Medical Applications: A New Look Inside Human Body With Raman Imaging; Solvent Suppression Methods in NMR Spectroscopy; Spectroscopic Methods in Drug Quality Control and Development; Spectroscopy for Process Analytical Technology (PAT); Spectroscopy in Biotechnology Research and Development; Structural Chemistry Using NMR Spectroscopy, Pharmaceuticals; UV-Visible Absorption Spectroscopy, Biomacromolecular Applications; Vibrational Spectroscopy Applications in Drugs Analysis.
Further Reading Albert K (1995) On-line use of NMR detection in separation chemistry. Journal of Chromatography A 703: 123–147. Albert K (1995) Direct on-line coupling of capillary electrophoresis and 1H NMR spectroscopy. Angewandte Chemie International Edition in English 34: 641–642. Albert K, Braumann U, Tseng L-H, et al. (1994) On-line coupling of supercritical fluid chromatography and proton high-field nuclear magnetic resonance spectroscopy. Analytical Chemistry 66: 3042–3046. Albert K, Kunst M, Bayer E, Spraul M, and Bermel W (1989) Reverse-phase highperformance liquid chromatography-nuclear magnetic resonance on-line coupling with solvent non-excitation. Journal of Chromatography 463: 355. Behnke B, Schlotterbeck G, Tallarek U, et al. (1996) Capillary HPLC-NMR coupling: High resolution NMR spectroscopy in the nanoliter scale. Analytical Chemistry 68: 1110–1115. Griffiths L (1995) Optimization of NMR and HPLC conditions for LC-NMR. Analytical Chemistry 67: 4091–4095.
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Laude DA Jr and Wilkins CL (1984) Direct-linked analytical scale high-performance liquid chromatography/nuclear magnetic resonance spectroscopy. Analytical Chemistry 56: 2471–2475. Lindon JC, Farrant RD, Sanderson PN, et al. (1995) Separation and characterization of components of peptide libraries using on-flow coupled HPLC-NMR spectroscopy. Magnetic Resonance in Chemistry 33: 857–863. Lindon JC, Nicholson JK, and Wilson ID (1996) Direct coupling of chromatographic separations to NMR spectroscopy. Progress in Nuclear Magnetic Resonance Spectroscopy 29: 1–49. Lindon JC, Nicholson JK, Sidelmann UG, and Wilson ID (1997) Directly coupled HPLC-NMR and its application to drug metabolism. Drug Metabolism Reviews 29: 705–746. Pullen FS, Swanson AG, Newman MJ, and Richards DS (1995) “On-line” liquid chromatography/nuclear magnetic resonance mass spectrometry—A powerful spectroscopic tool for the analysis of mixtures of pharmaceutical interest. Rapid Communications in Mass Spectrometry 9: 1003–1006. Shockcor JP, Unger SE, Wilson ID, Foxall PJD, Nicholson JK, and Lindon JC (1996) Combined HPLC, NMR spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine. Analytical Chemistry 68: 4431–4435. Shockcor JP, Wurm RM, Frick LW, et al. (1996) HPLC-NMR identification of the human urinary metabolites of (–)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine, a nucleoside analogue active against human immunodeficiency virus (HIV). Xenobiotica 26: 189–199. Smallcombe SH, Patt SL, and Keifer PA (1995) WET solvent suppression and its application to LC-NMR and high resolution NMR spectroscopy. Journal of Magnetic Resonance Series A 117: 295–303. Sweatman BC, Farrant RD, Sanderson PN, et al. (1995) Evaluation of the detection limits of directly coupled 600 MHz 1H and 1H-13C HPLC-NMR spectroscopy. Journal of Magnetic Resonance Analysis 1: 9–12. Wolfender J-L, Rodriguez S, and Hostettmann K (1998) Liquid chromatography coupled to mass spectrometry and nuclear magnetic resonance spectroscopy for the screening of plant constituents. Journal of Chromatography A 794: 299–316.