Melanie Voigt is grateful for a stipend from the Promotionskolleg of the Niederrhein University of Applied Sciences. The authors thank their institution for further financial support.

1. Sample Preparation: Solid Phase Extraction
<ol>
	<li>Collect about 1 L of water for the preparation of the samples.</li>
	<li>Filter the sample over a blue band filter with a pore size of 2 &#181;m to remove coarse particles.</li>
	<li>Equilibrate the SPE cartridge using 3 mL of methanol and 3 mL of ultrapure water.</li>
	<li>Apply the filtrate (1 L) onto the SPE cartridge and increase the flow velocity using a moderate vacuum, e.g., a diaphragm pump. 
	NOTE: Several SPE cartridges may be run in parallel.</li>
	<li>Wash the sample with 3 mL of ultrapure water.</li>
	<li>Elute the analytes from the cartridge sorbate with 3 mL of methanol.</li>
	<li>Concentrate the 3 mL eluate to dry using a rotary evaporator.</li>
	<li>Dissolve the residue in 1 mL of ultrapure water.</li>
	<li>Filter the solution through a syringe filter and store them in a vial for the non-targeted analysis by HPLC-ESI-Q-TOF-MS.</li>
</ol>
2. HPLC-ESI-Q-TOF-MS Method for Non-Targeted and Targeted Analysis, and MS/MS
<ol>
	<li>Transfer the vial to the HPLC-ESI-Q-TOF-MS autosampler.</li>
	<li>Set all relevant parameters (Table 1) for the HPLC-ESI-Q-TOF-MS. 
	NOTE: If a finite collision energy is used, i.e., collision energy (CE) &#8800; 0, ions will be fragmented. This mode corresponds to the targeted MS/MS method.</li>
	<li>Start the measurement.</li>
	<li>Analyze the resulting chromatograms and mass spectra.</li>
</ol>
3. UV Irradiation Experiments
<ol>
	<li>Dissolve the antibiotic compound, e.g., erythromycin (750 mg/L), in ultrapure water at 20 mg/L final concentration.</li>
	<li>Fill the 1 L photoreactor, wrapped in aluminum foil, with 750 mL of the solution.</li>
	<li>Introduce the lamp providing 15 W of power into the reactor.</li>
	<li>Apply the magnetic stirrer at 500 rpm.</li>
	<li>Adjust the pH value to the desired value 3-4, 6-7 or 8-9 by dropwise addition of HCl (0.1 M) or NH3 (0.1 M) if necessary. pH 6-7 is used as an example.</li>
	<li>Take 2 mL of the reaction solution as sample at time 0 using a syringe and transfer it into a 2 mL glass vial.</li>
	<li>Switch on the UV lamp and keep track of the elapsing time. 
	NOTE: Irradiation times of 10 min are often sufficient. If completeness of the photoreaction is desired, a second experiment series might need to be recorded using the results of the first series. 
	Caution: UV irradiation may lead to blindness.</li>
	<li>Draw a 2 mL sample from the solution every 30 s during the first 5 min. Then, take a sample every 60 s until the end of the experiment. Transfer the samples into 2 mL vials.</li>
	<li>Store the vials until HPLC-ESI-Q-TOF-MS analysis at -4 &#176;C.</li>
	<li>Analyze the 16 samples using HPLC-ESI-Q-TOF-MS with the methods described in step 2.</li>
</ol>
4. Kinetics Analysis
<ol>
	<li>Prepare a suitable software such as the curve-fitting toolbox of MATLAB R2016b.</li>
	<li>Fit the mass-area vs. time data of the photo-induced degradation of the parent antibiotic compound according to first order kinetics, see eq. 242,43 
	<img alt="Equation 1" src="/files/ftp_upload/57434/57434eq1.jpg" />&#160; &#160;(2) 
	The concentration <img alt="Equation 2" src="/files/ftp_upload/57434/57434eq2.jpg" /> refers to the initial concentration of the educt A, cA to the actual concentration over the reaction time t with the rate constant k1 from the first reaction step A to B.</li>
	<li>Fit the mass-area vs. time curves of the degradates using eq. 3 and 4, as they can be described as intermediates of a consecutive or subsequent follow-up reaction, i.e., product B or C according to the reaction model A &#8594;B &#8594; C &#8594; D. 
	<img alt="Equation 3" src="/files/ftp_upload/57434/57434eq3.jpg" />&#160; &#160;(3) 
	<img alt="Equation 4" src="/files/ftp_upload/57434/57434eq4v5.jpg" />&#160; &#160;(4) 
	The concentrations cB and cC refer to the intermediates B and C; and k2,k3 to the corresponding rate constants B to C, C to D.</li>
	<li>Use eq. 5 to fit the data, if the irradiation time was not sufficient to observe the degradation of a photo-product. This degradate can be treated as final product D with the concentration CD to obtain rate constants. 
	<img alt="Equation 6" src="/files/ftp_upload/57434/57434eq6.jpg" />&#160; &#160;(5)
	<ol>
		<li>Calculate the concentration of B using eq. 6 instead of eq. 3, if the reaction ends with B. If C is the final product, calculate the concentration of C according to eq. 7 instead of eq. 4. 
		<img alt="Equation 7" src="/files/ftp_upload/57434/57434eq7.jpg" />&#160; &#160;(6) 
		<img alt="Equation 8" src="/files/ftp_upload/57434/57434eq8.jpg" />&#160; &#160;(7)</li>
	</ol>
	</li>
	<li>Use eq. 8 for the determination of the half-lives t1/2. 
	<img alt="Equation 10" src="/files/ftp_upload/57434/57434eq10.jpg" />&#160; &#160;(8)</li>
</ol>

The authors declare no competing financial interests.

The example of a non-targeted analysis presented in this report demonstrated the identification of pharmaceuticals in surface water using HPLC-ESI-Q-TOF-MS, MS/MS and comparison with reference standards as the final proof. The strength of non-targeted analysis using TOF-MS is based on the detection of all ions present at a given retention time and the high mass accuracy which leads to the prediction of the tentative molecular formula. As an alternative to a TOF mass spectrometer, the application of an orbital ion trap has been described for contaminant analysis in water44. The molecular formula prediction was used as the starting point to quickly select reference standards. The application of the targeted MS method of the Q-TOF-MS instrument allowed the detection of specific compounds, since only pre-selected ions pass the quadrupole filter. In general targeted analysis is performed using triple quadrupole mass spectrometer also in water analysis45. To compensate for the deviation from the theoretical mass due to instrumental imperfections, a chromatographic comparison with a reference standard might be performed. The targeted MS/MS method may also be chosen for identification analysis. Here, ions are selected, fragmented and their fragments detected. Since MS/MS is less sensitive than MS, the concentration of the pharmaceuticals in the investigated water samples was too low to yield meaningful fragments. However, if fragments are detected, compounds can be identified with higher confidence. The insufficient sensitivity might be overcome by concentrating a larger initial water sample volume. In addition, the measurement should be carried out as soon as possible after sampling because of potential biodegradation46,47,48,49. Otherwise, samples should be stored at -20 &#176;C to exclude compound degradation or reaction.
Sometimes the same m/z values appear at different retention times. This may be due to that the isomers require different analytical techniques. It can also occur that no compounds might be detected at all, which does not necessarily prove their absence. They might just not form ions or occur below the limit of detection. The type of water also exercises an influence on the presence of pharmaceuticals. Pharmaceuticals rarely enter source water and groundwater as compared to sewage water and effluents from wastewater treatment plants48,50,51,52,53.
For the degradation experiments, the irradiation source should be characterized in advance, since the photon flux or photon fluence rate of the lamp contributes significantly to the degradation and the mechanism of degradation. For initial attempts, a VUV/UVC lamp, probably a low-pressure mercury lamp is sufficient. In general, the addition of hydrogen peroxide, H2O2, accelerates the degradation27,36,37,54. When a different lamp, e.g., a UVA lamp, is used, the formation of hydroxyl radicals should be ensured, e.g., through the addition of titanium dioxide 23,24,30,31. For many compounds, such as erythromycin, OH radicals rather than photo-reactivity of the pharmaceutical itself27are the degradation-inducing species.
For the determination of the kinetic parameters, the area of the signals in the mass-detected chromatograms, representing concentration, is plotted versus irradiation time. To fit the data, it is advisable to use suitable software. Here, the curve fitting tool of MATLAB was used, which allowed to quickly compute and fit the data with the correct equations. The kinetic of the intermediates is determined by more complex equations. The quality parameters for the fit, i.e., R2 and RMSE, were readily obtained as well.
This study demonstrated the analysis of river water to detect and identify pharmaceutical pollutants and the photodegradation of erythromycin in ultrapure water. In environmental waters, such as surface water, different degradation velocities and rate constants would be obtained due to light absorbing substances, such as humins. According to the authors&#39; experience, degradation often takes place more slowly, but sometimes at comparable rates41,56.
The worldwide problem of pharmaceuticals, especially antibiotics, in the aquatic environment and the resulting hazards still continue to grow1. Due to the variety and diversity of chemicals, metabolites, and degradates thereof, non-targeted analysis will become the most important analytical weapon for their discovery in the environment57. For the effective elimination, novel stages in wastewater treatment plants will need to be designed based on advanced oxidation processes, which UV irradiation might be part of.

Pharmaceuticals are regularly found in the aquatic environment1,2,3,4,5. An important source are effluents from wastewater treatment plants (WWTP)6,7,8,9. The occurrence of pharmaceuticals in the throughout the water cycle has been studied exemplarily in the Turia River Basin10. Among others, antibiotics represent a particular hazardous class of drugs, since they often pass the biological stage of WWTPs unaltered and may cause bacterial resistances in the environment11,12,13. Macrolides constitute a class of antibiotic drugs that are applied both in human and in veterinary medicine. Their representatives were found in concentration up to 1 &#181;g/L in effluents14,15,16,17,18,19. One of them is erythromycin (Ery)20,21. In waters, erythromycin is often accompanied by anhydroerythromycin A (Ery A - H2O), a dehydrate22,23. Water elimination from erythromycin is due to acid instability. The ratio of erythromycin vs. anhydroerythromycin depends on the pH24,25,26,27.
Chemically, macrolides contain a macrocylic lactone to which various sugar moieties are attached, e.g., desosamine, cladinose or mycaminose. Since macrolides are chemically modified natural products from fermentation processes, they often exist as mixtures. The species termed A, B, C, etc., differ in the sugar substituents. The sugar moieties and their position at the lactone are responsible for the mode of action of macrolides28,29. In order to minimize environmental hazard, it is desirable to completely mineralize the pharmaceuticals before entering the aquatic environment27,30,31,32.
The first part of this study deals with the detection of pharmaceutical in surface waters, which is important for monitoring both effluents and open waters. To search for a variety of unidentified substances in the microgram range in different matrices, non-targeted analysis is the method of choice20,33,34,35. In particular, high-performance liquid chromatography (HPLC) electrospray ionization quadrupole time of flight mass spectrometry (HPLC-ESI-Q-TOF-MS) has been proved of extraordinary value due to its specificity and sensitivity. After the identification of the substance, sensitivity can further be extended by using the targeted MS approach with the quadrupole operated in select mode and the collision energy within the collision cell set to zero. Hence, ions arrive non-fragmented at the TOF detector.
The second focus of this work is the elimination of erythromycin. For the elimination of the pharmaceuticals, so-called advanced oxidation processes (AOPs) are used, e.g., started by irradiation with UV light36,37,38. Essential for the degradation is the formation of hydroxyl radicals from water by VUV / UVC irradiation following eq. 1.
H2O + h&#957;(&#60;200 nm) &#8594; H2O* &#8594; H. + .OH&#160; &#160; &#160; &#160; (1)
Hydroxyl radicals possess a high oxidation potential of 2.8 V, which positively contributes to the degradation of the substances36,37.
Here, the degradation of erythromycin using vacuum UV/UVC-irradiation in water is described taking the influences of the pH into account. The formation of even more hazardous products is believed to be a disadvantage of using AOPs39,40. Thus, it is important to irradiate until complete mineralization of the pharmaceuticals. To better estimate the irradiation time, the kinetic model of the reaction, the reaction rate constants and the half-lives are determined both for the initial drug and for its photodegradates. To this purpose, concentration-time (c-t) plot were derived from HPLC-ESI-Q-TOF-MS measurements and compared to chemical kinetics models using MATLAB. The kinetics of the degradation proceeded according to first-order, and the photodegradates were described as intermediate products of a consecutive or subsequent follow-up reaction27,41.

<table><tbody><tr><td>Methanol for liquid chromatography LiChrosolv</td><td>Merck</td><td>1060181000</td><td></td></tr><tr><td>formic acid</td><td>Fluka</td><td>94318</td><td></td></tr><tr><td>HCl</td><td>Riedel-de Haen</td><td></td><td></td></tr><tr><td>NH3</td><td>Riedel-de Haen</td><td></td><td></td></tr><tr><td>Simplicity 185 Water Purification System</td><td>EMD Millipore</td><td></td><td>for producing MilliQ-water</td></tr><tr><td>Erythromycin</td><td>BioChemica AppliChem</td><td>A2275,0005</td><td></td></tr><tr><td>Filter Rotilabo-filter, Typ 113A</td><td>Roth</td><td>AP78.1</td><td></td></tr><tr><td>SPE-Cartridges Oasis HLB 3cc (60mg)</td><td>Waters</td><td>WAT094226</td><td></td></tr><tr><td>BAKER SPE-12G</td><td>J.T. Baker</td><td></td><td></td></tr><tr><td>membrane pump PC3001 VarioPro&amp;nbsp;</td><td>Vacuubrand</td><td></td><td></td></tr><tr><td>rotary evaporator; Laborota 4000 efficient</td><td>Heidolph Instruments</td><td></td><td></td></tr><tr><td>syringe, 2 mL</td><td>Terumo</td><td></td><td></td></tr><tr><td>Nylon Syringe Filters Target2</td><td>Thermo Scientific</td><td>10301345</td><td></td></tr><tr><td>C-18 CoreShell column 50 mm x 2.1 mm dimensions, 2.6 &amp;mu;m particle size</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>HPLC 1200</td><td>Agilent</td><td></td><td></td></tr><tr><td>ESI-Q-ToF-MS 6530</td><td>Agilent</td><td></td><td></td></tr><tr><td>photoreactor, UV Labor Reactor System 3</td><td>Peschl Utraviolet GmbH</td><td></td><td></td></tr><tr><td>VUV/UVC-lamp, TNN 15/32, 15 W</td><td>Heraeus</td><td></td><td></td></tr><tr><td>pH-meter, pHenomenal pH 1100L</td><td>vwr</td><td>662-1657</td><td></td></tr><tr><td>magnetic stirrer</td><td>Heidolph Instruments</td><td></td><td></td></tr><tr><td>MassHunter Workstation B.06.00</td><td>Agilent</td><td></td><td></td></tr><tr><td>MATLAB R2016b</td><td>Mathworks</td><td></td><td></td></tr></tbody></table>

identification of pharmaceuticals in the aquatic environment using hplc-esi-q-tof-ms and elimination of erythromycin through photo-induced degradation

Monitoring pharmaceuticals throughout the water cycle is becoming increasingly important for the aquatic environment and eventually for human health. Targeted and non-targeted analysis are today's means of choice. Although targeted analysis usually conducted with the help of a triple quadrupole mass spectrometer may be more sensitive, only compounds previously selected can be identified. The most powerful non-targeted analysis is performed through time of flight mass spectrometers (TOF-MS) extended by a quadrupole mass analyzer (Q), as used in this study. Preceded by solid phase extraction and high-performance liquid chromatography (HPLC), the non-targeted approach allows to detect all ionizable substances with high sensitivity and selectivity. Taking full advantage of the Q-TOF-MS instrument, tandem mass spectrometry (MS/MS) experiments accelerate and facilitate the identification while a targeted MS method enhances the sensitivity but relies on reference standards for identification purposes. The identification of four pharmaceuticals from Rhine river water is demonstrated. The Rhine river originates in Tomasee, Graub&#252;nden, Switzerland and flows into the North Sea, near Southern Bight, The Netherlands. Its length amounts to 1232.7 km. Since it is of prime interest to effectively eliminate pharmaceuticals from the water cycle, the effect UV-C irradiation is demonstrated on a laboratory scale. This method allows fast degradation of pharmaceuticals, which is exemplarily shown for the macrolide antibiotic erythromycin. Using the above HPLC-Q-TOF-MS method, concentration-time diagrams are obtained for the parent drug and their photodegradation products. After establishing the equations for first-order sequential reactions, computational fitting allows the determination of kinetic parameters, which might help to predict irradiation times and conditions when potentially considered as fourth stage within wastewater treatment.

As result of the solid phase extraction, a yellowish to dark green solution was obtained in all cases, which indicated the presence of chlorophyll-containing substances (Figure 1). Pharmaceuticals contained in this water sample would not lead to visible coloration since their concentration and their absorbance would generally be too low. Instead, the occurrence of pharmaceuticals needs to be analyzed using HPLC and high-resolution mass spectrometry.
In non-targeted analysis, a HPLC-ESI-Q-TOF-MS was used because of its outstanding mass accuracy allowing to obtain the accurate mass for each compound ion. The mass-detected chromatogram of the performed analysis was represented as a base peak chromatogram (BPC), which displays the most intense peak of each mass spectrum recorded in the course the chromatographic separation. The example shown in Figure 2 presents the BPC of a water sample from the river Rhine.
The BPC contained more than twenty-five peaks reflecting different m/z values, hence different compounds, seven of which were marked in the BPC. Since the substances were unknown a priori, the first step to their identification usually consists of deriving the molecular formula. This is accomplished through accurate mass and isotopic pattern provided by the TOF detection, although the isotopic pattern may not be observed in all cases due to low sample concentrations in environmental samples. With the help of public database, such as Pharmaceuticals in the Environment by the German Environment Agency (UBA) containing approximately 630 compounds, a preliminary identification of a small group of candidates is often successful. For a final proof, either comparison to commercially available reference standards may be performed or MS/MS fragmentation patterns may be considered (Figure 3).
In this work, comparison to standards with respect to retention time accounted for the identification of pharmaceuticals very often found in German surface waters. These substances include metoprolol, a &#946;-blocker, carbamazepine, an analgesic, and the macrolide antibiotic erythromycin A and its derivate anhydroerythromycin A. Erythromycin serves as example further investigated in this study. The studied Rhine river sample had a pH of 7.6 and an average temperature of 16.5 &#176;C. At this pH, anhydroerythromycin would also be expected to be present in the water sample. For the detailed analysis, the extracted ion chromatograms (EICs) of the water sample were compared with the reference standards (Figure 4).
The comparison shows good agreement between the retention time for metoprolol, carbamazepine and anhydroerythromycin and the observed analytes. The EIC of the reference standard anhydroerythromycin displayed two peaks, hence two compounds where dehydration had occurred at two distinct sites of erythromycin. Yet, only one anhydroerythromycin isomer was identified in the Rhine river sample. Erythromycin itself was only present in traces. Therefore, no MS/MS spectrum could be obtained. The accurate masses for the antibiotic and its dehydrate are given in Table 2. Using EIC, thus m/z value and retention time, metoprolol, carbamazepin, erythromycin and anhydroerythromycin could be identified in the Rhine river sample.
With respect to the aquatic environment, it is important to prevent pharmaceuticals from passing through wastewater treatment plants and entering surface waters. In the quest for an efficient elimination, UV-C irradiation experiments at different pH values were carried out for erythromycin as example. Concentration-time (c-t) diagrams were recorded using mass-area vs. time plots derived from EICs. The degradation was described according to equation 2. Erythromycin consists of erythromycin A and B and anhydroerythromycin A, with two isomers of the latter. The c-t curves of erythromycin A and their computational fits are shown in Figure 5. At pH 7,accelerated degradation was observed. This applies to all four compounds studied, data not shown. As a consequence, photo-induced degradation of erythromycin should be carried out around neutral pH. In the case of the Rhine river sample, pH adjustment was not required.
Photodegradates of the pharmaceuticals were also identified at all three pH values. An overview of these photodegradates with their corresponding structure proposals is given in Table 3. For the kinetic analysis of the photodegradates, the product with m/z = 720 serves as an example. Photodegradates can often be described as reaction intermediates. Therefore, the photodegradates were described in terms of aconsecutive and subsequent follow-up reaction. The decision between the resulting types of intermediates is based on the goodness of the fit computed with suitable software, where the coefficient of determination (R2) and the residual mean-squared error (RMSE) were taken as the criteria. Due to the fact that erythromycin is acid-instable, degradates as would occur upon irradiation were present prior to irradiation. The resulting effect on equations 3 and 4 was a finite starting concentration. Hence, a factor was added to the equations. Figure 6 shows experimental data and fits computed according to equation 3 and 4.
This example of an intermediate demonstrated the concentration increase with a sigmoidal rise followed by an exponential decay. This is indicative for a subsequent follow-up reaction intermediate. A consecutive reaction intermediate does not show the sigmoidal increase. Statistical quality parameters also indicated the slightly superior agreement of fit according to the subsequent follow-up reaction model. The coefficient of determination R2 of the consecutive reaction was 0.9898 and thus lower than that of the subsequent follow-up reaction being 0.9976. Therefore, the examined photoproduct was interpreted as intermediate of a subsequent follow-up reaction. The k-values resulted from the computational fit as well, the half-life was calculated following equation 5. All relevant kinetic parameters are collected in Table 3.
The fastest degradation was observed at pH 7, followed by pH 9, while the slowest degradation was found for pH 3 (Figure 5). This finding also applied to the formation and degradation of the photoproducts. Three photodegradates were observed. Their m/z values were 750.46 corresponding to Ery F, 720.45 to Ery C and 192.12 to DPEry192, a glycosidically bound sugar of the erythromycin structure (Figure 7). No degradation of the photoproduct could be observed for DPEry192 at pH 3 and 9 and for Ery F at pH 9. In these cases, the irradiation time was not sufficient long to observe total degradation of the intermediate product. Nevertheless, the formation rate constant could be determined by using equation 5, which corresponds to a final product.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57434/57434fig1.jpg" /> 
Figure 1. Comparison of the samples from the Rhine river after SPE (left) and ultrapure water(right) treatment. The green coloration is indicative for chlorophyll-containing substances. <a href="/files/ftp_upload/57434/57434fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57434/57434fig2.jpg" /> 
Figure 2. BPC of a water sample after SPE measured with HPLC-ESI-Q-TOF-MS. All chromatograms were normalized to the highest peak. Illustrative m/z-values as obtained from the corresponding MS spectrum are marked. <a href="/files/ftp_upload/57434/57434fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57434/57434fig3.jpg" /> 
Figure 3. Q-TOF-MS spectrum of erythromycin A (bottom) and the MS/MS spectrum of the ion m/z = 734.4689 (top). The spectra show the quasi-molecular ion of erythromycin A with its isotopic pattern and the fragments at an applied collision energy of 30 eV. <a href="/files/ftp_upload/57434/57434fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57434/57434fig4.jpg" /> 
Figure 4. Normalized EICs of (A) metoprolol, (B) carbamazepine, (C) erythromycin A and (D) anhydroerythromycin A in a Rhine river sample (blue) and in ultrapure water from reference compounds (red). The retention times of the reference compounds and the ones of the pharmaceuticals in the water sample are the same. The signal-to-noise ratios of metoprolol (A) and anhydroerythromycin (D) are higher than those of carbamazepine (B) and erythromycin (C), which indicates the latter were present only in traces. <a href="/files/ftp_upload/57434/57434fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57434/57434fig5.jpg" /> 
Figure 5. Normalized concentration-time curves of the photodegradation of erythromycin A at pH 3 (red), pH 7 (green) and pH 9 (blue). Solutions were irradiated for 10 min. At pH 7, erythromycin was completely removed from the sample. The concentration-time curves could be described using first-order kinetic equations. The kinetic rate constants were 0.10 (pH 3), 0.59 (pH 7) and =0.21 (pH 9). <a href="/files/ftp_upload/57434/57434fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57434/57434fig6.jpg" /> 
Figure 6. Comparison of the fits of the concentration-time curves of the photoprodegradates of erythromycin with m/z = 720 at pH 9 following equations 3 (A) and 4 (B). Goodness of the fit of the consecutive reaction (A): R2 = 0.9898, RMSE = 4.645E+04, and of the subsequent follow-up reaction (B): R2 = 09976, RMSE = 2.366E+04. <a href="/files/ftp_upload/57434/57434fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57434/57434fig7.jpg" /> 
Figure 7. Structure of erythromycin A, erythromycin B and anhydroerythromycin and their photdegradation products. This figure has been modified from Voigt et al.27. The products were formed after 10 min of UVC-irradiation and identified using HPLC-Q-TOF-MS and MS/MS. <a href="/files/ftp_upload/57434/57434fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Liquid Chromatography</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Column:</td>
			<td colspan="6">reversed-phase C-18</td>
		</tr>
		<tr>
			<td>Column:</td>
			<td colspan="6">CoreShell column;</td>
		</tr>
		<tr>
			<td>Column:</td>
			<td colspan="6">50 mm x 2.1 mm dimensions, 2.6 &#956;m particle size</td>
		</tr>
		<tr>
			<td>Column temperature</td>
			<td colspan="6">40 &#176;C</td>
		</tr>
		<tr>
			<td>Injection volume:</td>
			<td colspan="6">5 &#181;L</td>
		</tr>
		<tr>
			<td>Flow:</td>
			<td colspan="6">0.3 mL/min</td>
		</tr>
		<tr>
			<td>Mobile phase:</td>
			<td colspan="6">Solvent A: water containing 0.1% formic acid</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="6">Solvent B: methanol containing 0.1% formic acid</td>
		</tr>
		<tr>
			<td>Gradient program:</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Time /min</td>
			<td>0</td>
			<td>1</td>
			<td>10</td>
			<td>11.1</td>
			<td>11.2</td>
			<td>12</td>
		</tr>
		<tr>
			<td>A:B solvent ratio</td>
			<td>99:1</td>
			<td>70:30</td>
			<td>25:75</td>
			<td>1:99</td>
			<td>1:99</td>
			<td>99:1</td>
		</tr>
		<tr>
			<td>Mass Spectrometry</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Source:</td>
			<td colspan="6">Dual AJS ESI (positive mode)</td>
		</tr>
		<tr>
			<td>Gas and source</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Gas Temperature:</td>
			<td colspan="6">300 &#176;C</td>
		</tr>
		<tr>
			<td>Drying Gas:</td>
			<td colspan="6">8.0 L/min</td>
		</tr>
		<tr>
			<td>Nebulizer:</td>
			<td colspan="6">14 psig</td>
		</tr>
		<tr>
			<td>Sheath Gas Temperature:</td>
			<td colspan="6">300 &#176;C</td>
		</tr>
		<tr>
			<td>Sheath Gas Flow:</td>
			<td colspan="6">8 L/min</td>
		</tr>
		<tr>
			<td>Mass Range:</td>
			<td colspan="6">100 - 1000 m/z</td>
		</tr>
		<tr>
			<td>Acquisition Rate:</td>
			<td colspan="6">1 spectrum/s</td>
		</tr>
		<tr>
			<td>Acquisition Time:</td>
			<td colspan="6">1000 ms/spectrum</td>
		</tr>
		<tr>
			<td>Transient/ spectrum</td>
			<td colspan="6">10014</td>
		</tr>
		<tr>
			<td>For targeted MS method</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Collision energy (CE):</td>
			<td colspan="6">0 eV</td>
		</tr>
		<tr>
			<td>Preferred Mass - Table</td>
			<td colspan="6">734.4685</td>
		</tr>
		<tr>
			<td>For MS/MS (typically auto MS/MS mode)</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Collision energy (CE):</td>
			<td colspan="6">30 eV</td>
		</tr>
		<tr>
			<td>Absolute threshold</td>
			<td colspan="6">3000 counts</td>
		</tr>
		<tr>
			<td>Relative threshold</td>
			<td colspan="6">0.01 %</td>
		</tr>
		<tr>
			<td>Mass Range:</td>
			<td colspan="6">100 - 100 m/z</td>
		</tr>
		<tr>
			<td>Acquisition Rate:</td>
			<td colspan="6">1 spectrum/s</td>
		</tr>
		<tr>
			<td>Acquisition Time:</td>
			<td colspan="6">1000 ms/spectrum</td>
		</tr>
		<tr>
			<td>Transient/ spectrum</td>
			<td colspan="6">9964</td>
		</tr>
		<tr>
			<td>For targeted MS/MS method</td>
			<td colspan="6"></td>
		</tr>
		<tr>
			<td>Preferred Mass - Table</td>
			<td colspan="6">734.4685</td>
		</tr>
	</tbody>
</table>
Table 1. Conditions and parameters used for HPLC-ESI-Q-TOF-MS analysis of pharmaceuticals in water matrices. It is advisable to introduce a rinsing step between the chromatographic runs through running a sample of pure ultrapure water between two analyses or through extending the run time of the chromatographic method in order to elute all substances.
<img alt="Table 2" src="/files/ftp_upload/57434/57434table2.jpg" /> 
Table 2. Pharmaceuticals found in the Rhine river sample with their retention time, theoretical and observed [M+H]+ and their structure. The ESI mode was set to positive, so that [M+H]+-ions were detected. The retention time may vary minimally for usual experimental known reasons.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>pH 3</td>
			<td>pH 3</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 7</td>
			<td>pH 9</td>
			<td>pH 9</td>
			<td>pH 9</td>
			<td>pH 9</td>
		</tr>
		<tr>
			<td>Product</td>
			<td>k1 [min-1]</td>
			<td>t1/2 [min] (k1)</td>
			<td>k1 [min-1]</td>
			<td>k2 [min-1]</td>
			<td>k3 [min-1]</td>
			<td>t1/2 [min] (k1)</td>
			<td>t1/2 [min] (k2)</td>
			<td>t1/2 [min] (k3)</td>
			<td>k1 [min-1]</td>
			<td>k2 [min-1]</td>
			<td>t1/2 [min] (k1)</td>
			<td>t1/2 [min] (k2)</td>
		</tr>
		<tr>
			<td>Ery A&#160;</td>
			<td>0.1</td>
			<td>6.81</td>
			<td>0.59</td>
			<td>-</td>
			<td>-</td>
			<td>1.18</td>
			<td>-</td>
			<td>-</td>
			<td>0.21</td>
			<td>-</td>
			<td>3.37</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery B&#160;</td>
			<td>0.05</td>
			<td>14.23</td>
			<td>0.66</td>
			<td>-</td>
			<td>-</td>
			<td>1.04</td>
			<td>-</td>
			<td>-</td>
			<td>0.22</td>
			<td>-</td>
			<td>3.21</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery A &#8211; H2Oa&#160;</td>
			<td>0.11</td>
			<td>6.53</td>
			<td>0.59</td>
			<td>-</td>
			<td>-</td>
			<td>1.17</td>
			<td>-</td>
			<td>-</td>
			<td>0.19</td>
			<td>-</td>
			<td>3.72</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery A &#8211; H2Ob</td>
			<td>0.15</td>
			<td>4.76</td>
			<td>1.11</td>
			<td>-</td>
			<td>-</td>
			<td>0.63</td>
			<td>-</td>
			<td>-</td>
			<td>0.21</td>
			<td>-</td>
			<td>3.35</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery F&#160;</td>
			<td>not observed</td>
			<td>-</td>
			<td>0.89</td>
			<td>0.35</td>
			<td>-</td>
			<td>0.78</td>
			<td>1.98</td>
			<td>-</td>
			<td>1.09*</td>
			<td>-</td>
			<td>0.64</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ery C</td>
			<td>not determined</td>
			<td>-</td>
			<td>0.74</td>
			<td>5.27</td>
			<td>0.78</td>
			<td>0.94</td>
			<td>0.13</td>
			<td>0.89</td>
			<td>0.17</td>
			<td>0.18</td>
			<td>4.04</td>
			<td>3.92</td>
		</tr>
		<tr>
			<td>DPEry192</td>
			<td>0.35*</td>
			<td>1.97</td>
			<td>not observed</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>0.30*</td>
			<td>-</td>
			<td>2.34</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="13">* No further degradation observed</td>
		</tr>
	</tbody>
</table>
Table 3. Kinetic rate constants and corresponding half-lives of the degradation of erythromycin and its photodegradates adapted from Voigt et al.27. Erythromycin consists of erythromycin A, erythromycin B and two forms of anhydroerythromycin. Three photodegradates were observed. There are referred to as Ery F, Ery C and DEry192.

Watch this Scientific Journal Video about Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation at JoVE

Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation

We present a protocol for non-targeted analysis using time of flight mass spectrometry as a perfect tool to identify pharmaceuticals in waters. We demonstrate the application of UV irradiation for their elimination. Analysis involving irradiation, compound isolation, identification and kinetic modelling of the degradation profiles is illustrated.

Monitoring pharmaceuticals throughout the water cycle is becoming increasingly important for the aquatic environment and eventually for human health. ...

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Research

JoVE Journal

Environment

13.0K Views.  Niederrhein University of Applied Sciences. Presenteren we een protocol voor niet-gerichte analyse met behulp van tijd van de Spectrometrie van de massa van de vlucht als een perfect hulpmiddel om te identificeren van farmaceutische producten in wateren. We laten zien dat de toepassing van UV bestraling voor hun verwijdering. Analyse waarbij bestraling, samengestelde isolatie, identificatie en kinetische modellen van de profielen van de afbraak wordt geïllustreerd.

Video: Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 &#177; 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

An improved polymerase chain reaction-restriction fragment length polymorphism method for genotyping pufferfish species by liquid chromatography/mass spectrometry is described. A reverse-phase silica monolith column is employed for separating digested amplicons. This method can elucidate the monoisotopic masses of oligonucleotides, which is useful for identifying base composition.

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

Genetics

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Biochemistry

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Chemistry

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Enhanced Isomeric Separation of Ergot Alkaloids Using LC-IMS-MS

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules).
Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence.
Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Biology

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation

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